Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs.Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In  In this study, Lawlor et al. show that transposons on the Drosophila Y chromosome have evolved to exploit a specific stage of spermatogenesis when the Y chromosome is decondensed and the piRNA pathway is downregulated. On the other hand, it is also possible that, like many viruses, TEs are engaged in an evolutionary arms race with their hosts, with TEs continuously evolving to escape silencing and the host genome continuously evolving to reestablish TE suppression4. Many host genes involved in TE defense are rapidly evolving, consistent with ongoing host-TE conflict9, however relatively few strategies where TEs can escape or evade host silencing have been identified10. In the Drosophila ovary, there is evidence that some TEs propagate in permissive nurse cells and hijack the host’s mRNA transport pathway to move to the developing oocyte, which is more recalcitrant to TE expression11. In another study, Dufourt et al. identified a small region of mitotically dividing germline cysts where the piRNA pathway effector protein Piwi is depleted and TE silencing is much weaker than in the surrounding cells. They termed this region the “piwiless pocket” and proposed that TEs may take advantage of this niche to replicate in the Drosophila germline13.Transposable elements (TEs) are abundant in the genomes of plants and animals despite the presence of sophisticated host genome defense pathways. The genetic mechanisms responsible for the evolutionary success and persistence of TEs remain unclear. It is possible that the fitness benefit of complete TE suppression is not large enough to be evolutionarily favorableDrosophila ovary, however surprisingly little is known about these same phenomena in the testes. Several previous observations suggest that there may be substantial differences between ovaries and testes with respect to both TE activity levels and host silencing pathways. For example, the expression patterns of multiple TE families are known to exhibit strong sex biases: The I-element, P-element, and gypsy TE families are all expressed at higher levels in the female germline16, whereas the opposite is true for the copia, micropia, 1731, and 412 TE families20. The piRNA pathway is active in both somatic and germline cells in the ovary and piRNAs bound by Aub and Ago3 undergo robust ping-pong amplification in the ovarian germline. In the testes, TE-derived piRNAs are produced in germ cells, however the vast majority (~75%) arise from the suppressor of stellate [Su(Ste)] and AT-chX satellite repeats, rather than the canonical piRNA clusters that have been identified in ovaries22. Furthermore, many TE families show large differences in piRNA abundance between ovaries and testes22 and TE-derived piRNAs only show a weak signature of ping-pong amplification in spermatocytes, likely due to low levels or absence of Ago321.TE replication and host silencing have been extensively studied in the Drosophila testes. Our method for identification of TE and host gene co-expression modules shows that a subset of primary spermatocytes expresses a diverse group of TEs at high levels relative to other cell types. These TEs are co-expressed with Y-linked fertility factors and we find evidence that they are more active in males compared to females. These data suggest some TEs may exploit spermatocyte-specific transcriptional programs and Y-chromosome activation to remain active in the Drosophila melanogaster genome.Here, we analyze TE expression at single-cell resolution to gain insight into the dynamics of TE activity in D. melanogaster strain w111823. The Drosophila larval testes are elongated spheres encased in epithelial cells. The cell types of the testes are ordered spatially along a developmental gradient: The apical cap contains germline stem cells and somatic hub cells. The germline stem cells give rise to daughter cells that undergo mitosis with incomplete cytokinesis to create cysts containing 16 spermatogonial cells. Each cyst is encapsulated by somatic cyst cells and the spermatogonial cells further differentiate to form meiotic spermatocytes. In the larval testes, the primary spermatocytes exist in an extended meiotic prophase and do not undergo further differentiation.We reanalyzed 10× Genomics 3′ single-cell expression data from a recent study examining sex chromosome gene expression in larval testes from We clustered cells based on their gene expression profiles to create cell clusters Fig.  and assi23, we identify distinct somatic and germline clusters  are defined largely by Fas3 expression, and pigment cells (cluster 9) express Sox100B  represent the remaining filtered cells Fig.  and exprm25 Fig. . These cm25 Fig. . Mean exm25 Fig. . Taken tDrosophila testis . We then visualized cell-type-specific expression patterns of all TEs with at least three UMIs detected across all individual cells in the tis Fig. . Most sttis Fig. . In comptis Fig. . Four antis Fig. . Interestis Fig.  and thertis Fig. , B. Nota23. TE expression levels from the bulk RNA-seq data are highly concordant with pseudobulk levels from the scRNA-seq data, both globally and with respect to TEs specifically . Only a small number of TEs show evidence of reproducible chimeric transcripts  has previously been shown to have this property and, in general, it performs favorably compared to other module detection methods28 and selected parameters that resulted in modules representing a wide range of biological processes while also showing minimal overlap in terms of their gene content (see Methods). The modules resulting from this approach were reproducibly identified across replicate runs of consensus ICA  term not enriched in any other module  included over 70 transposons along with approximately 300 host genes  for module 27 on the UMAP projection and observed that it is expressed mainly by cells in cluster 3 and a small number of cluster 2 cells, in agreement with our visual inspection of TE expression across the dataset   in cluster 3. Testis-specific Meiotic Arrest Complex (tMAC) components aly and wuc, which promote transcription of spermatocyte-specific genes by activating alternative promoters33, are members of module 27, as well as kmg, which blocks promiscuous activation of genes by tMAC34. Each of these is also significantly differentially expressed in cluster 3  .GO enrichment analysis shows that module 27 is enriched for genes that function in axonemal assembly and cilium movement, including the Y-chromosome fertility factors 36. Y-chromosome lampbrush loops also form at this stage of development and the Y chromosome becomes enriched for the H3K9ac histone modification, which is associated with active transcription37. Consistent with this phenomenon, we also find that tplus3a and tplus3b, two genes required for expression of Y-chromosome fertility factors38, are members of module 27 as well as bol, which binds the decondensed giant introns of several Y loop-forming genes39. All nine genes , are differentially expressed  in cluster 3 spermatocytes  of module 27 TEs have at least one full-length copy located on a known Y-linked scaffold  single-nucleotide variants in module 27 TEs. We then compared the relative abundance of each male-specific variant in the w1118 ribosomal RNA-depleted bulk RNA-seq data from adult testes to its relative abundance in male WGS data (see Methods). A ratio larger than 1 indicates the presence of one or more Y-linked TE insertions that are expressed more highly than total-copy number alone would explain. For each expressed TE, we found the site of the male-specific allele most overexpressed relative to WGS depth. At these sites, male-specific module 27 TE alleles have significantly higher ratios of relative RNA to DNA coverage compared to male-specific alleles from TEs not present in module 27    to compare insertion frequencies of module 27 TEs versus other active TEs. We found that other TEs exhibit similar X and autosomal insertion rates across the DGRP lines whereas module 27 TEs exhibit a significantly reduced frequency of X-linked insertions relative to autosomal insertions, consistent with male-biased activity  of the TEs in module 27 have at least one full-length copy within the assembled portion of the Y chromosome and/or are flamenco-regulated. This is likely an underestimate given estimates that ~60% of the Y chromosome remains unassembled40.Notably, not all TE families with intact Y-linked insertions are members of module 27, suggesting that additional features beyond Y-linkage, such as specific regulatory elements, are required for TEs to exploit this opportunity. Four module 27 TEs, Flamenco is not unique in terms of its female-biased piRNA production—the majority of known piRNA clusters produce more abundant piRNA in ovaries compared to testes22. Spermatocytes also lack a robust ping-pong amplification loop and the bulk of spermatocyte piRNAs come not from TEs, but rather two satellite repeats: su(Ste) and AT-chX57. Furthermore, piRNA factors such as Piwi and Ago3, while abundant in germline stem cells and spermatogonia, are missing or present at low levels in spermatocytes57. Our analysis of scRNA-seq data confirm these findings  represent a cryptic meiotic drive system where X-linked stellate genes disrupt spermatogenesis and cause sex-ratio distortion in the absence of the Y-linked su(Ste) piRNA cluster61. The function of AT-chX is less clear. Although this locus was originally proposed to play a role in the developmental silencing of vasa during spermatogenesis62, recent results instead suggest of role for AT-chX in hybrid incompatibility63. Neither locus is present in the genomes of close relatives of D. melanogaster, suggesting that they are dispensable for spermatogenesis64. The fact that both su(Ste) and AT-chX rapidly evolved to be essential for fertility in D. melanogaster is consistent with a role in mediating genetic conflict. This is especially clear for su(Ste) where the stellate protein is completely absent from wild-type flies64. If the su(Ste) and AT-chX piRNAs evolved to supress segregation distorters or other forms of selfish elements, it would suggest that there has been a tradeoff in the piRNA system in D. melanogaster spermatocytes, where increased abundance of su(Ste) and AT-chX piRNAs comes at a cost of impaired TE silencing.The downregulation of the piRNA pathway in spermatocytes is surprising. One would expect the piRNA pathway to be upregulated in these cells to safeguard against TE insertions resulting from de-repression of Y-linked TEs. So, why do spermatocytes show a weakened piRNA response at a developmental timepoint when the TE-rich Y chromosome is de-repressed? One possibility is related to intragenomic conflict. Sex chromosomes are hotspots for genomic conflictmelanogaster, D. simulans testes also show a weakening of piRNA-mediated TE silencing. D. simulans testes have been shown to have many fewer TE-derived piRNAs and higher TE expression compared to ovaries65. However, D. simulans lacks both the su(Ste) and AT-chX satellites. Thus, either the tradeoff between satellite- and TE-derived piRNAs is unique to D. melanogaster or a different tradeoff is present in D. simulans, such as between piRNA production versus other small RNAs. For example, siRNAs generated by hairpin RNAs have recently been shown to play an important role in resolving intragenomic conflict in the D. simulans male germline59. Alternatively, there may be no tradeoff at all and the downregulation of the piRNA pathway during spermatogenesis may serve some other, as yet undetermined, developmental function.Like Drosophila. Our method for identifying gene/TE co-expression modules allowed us to identify a TE-rich expression module that is active in early spermatocytes. These results suggest that spermatocytes represent a permissive niche for TE expression due to the downregulation of piRNA pathway components and the transcriptional activation of the TE-rich Y chromosome in these cells. Additional work to improve the Y-chromosome assembly and uncover the mechanisms underlying the transcriptional activation of the Y chromosome will be critical for understanding the burst of TE expression that we have identified in spermatocytes. Furthermore, future work investigating the peculiarities of TE silencing in the testes will help shed light upon the tradeoffs and constraints imposed by the various roles of piRNAs in the male germline, including host gene regulation, TE silencing, and the resolution of intragenomic conflicts.In summary, this work provides detailed insight into the expression dynamics of TEs in the male germline of 66 with the following options: “-e ncbi -s -no_is -nolow.” We used RepBase D. melanogaster consensus TE sequences (version 20170127)67 as a custom library.All repeat masking was performed with RepeatMaskerD. melanogaster r6.22 genome assembly.All Illumina read alignments were performed using a custom reference sequence made by appending the consensus TE sequences to the TE-masked PRJNA548742 and PRJNA518743 with wget v1.1468. We used 10X Genomics cellranger software to align and quantify the data69. We generated a cellranger index from the previously described custom reference sequence using cellranger’s “mkref” command with default parameters. We aligned scRNA-seq reads using cellranger’s “count” command with default parameters. We used cellranger’s filtered count matrices for further analysis.Single-cell RNA-seq data was downloaded from 70 v0.2.1 to these unnormalized count matrices to identify and filter putative heterotypic doublets. We used scanpy71 v1.6.0 to retain genes detected in at least 3 cells and then cells with at least 250 and fewer than 5000 detected genes. We removed cells with more than 5% of remaining UMIs assigned to mitochondrion-encoded genes. We normalized UMI counts to 10000 per cell and applied log transformation with a pseudo-count of 1. We generated pseudobulk expression estimates by summing post-filtering counts across all cells and assessed the concordance of these normalized values with expression in four larval testis poly-A selected RNA-seq replicates23  suffer from stochastically varying solutions. Kotliar et al. have previously introduced an elegant approach, termed consensus NMF (cNMF), to stabilize NMF solutions for scRNA-seq GEP detection28. This approach clusters the results of many iterations of NMF to buffer the influence of outlier solutions yielded by single runs of the algorithm. However, when we implemented this approach, we found that it yielded large GEPs utilized by broad cell types. We therefore chose to use ICA factorization because this approach was able to group genes into smaller GEPs expressed specifically by smaller cell populations.We chose ICA to identify gene expression programs because it performs highly with respect to recovering known functional gene modules and because it is easily adaptable to finding partially overlapping modules(k), our approach uses FastICA via sklearn74 to decompose the standardized expression matrix 100 times, then concatenates the resulting gene x module matrices, partitions all modules into k clusters using k-means clustering, and averages the per-cell scores within each partition to yield a consensus cell x module matrix. The values in this matrix represent the expression of each module in each cell and we refer to these values as module expression scores. For the same partitions, we averaged per-gene scores from cell by module matrices to generate a consensus cell by module matrix. These values represent the strength of the evidence for co-expression of a given gene/TE with the other features in the module. We refer to these values as module membership scores.We applied a consensus approach similar to Kotliar et al. to ICA to address the issue of ICA solution randomness. We performed ICA factorization using expression values for highly variable genes identified by scanpy as well as the 128 TE families that passed initial filtering. Therefore, resulting modules may contain exclusively TEs or genes or a mix of both types of feature. We standardized the normalized, log-transformed expression matrix containing the highly variable genes identified by scanpy and TEs to have zero mean and unit variance. Standardized scores were clipped to a maximum absolute value of 10. Then, for any desired number of components 75 to the vector of gene weights for each module. Genes with FDR q-values less than a desired cutoff (q) for each module were considered members of the module.We assigned genes to each program by applying fdrtool(k and q) used for this approach in an unbiased way, we applied a grid search approach to choose the two most important parameters of our pipeline (see Module Detection Parameters below).To obtain the two major parameters P < 0.05) with a module in all grid search replicate runs. . We found that all main result modules were significantly positively correlated  to request from the decomposition algorithm. Additionally, generation of discrete gene lists for each gene program requires application of arbitrary score cutoffs to determine program membership for each gene.Use of ICA or other matrix decomposition approaches for gene program detection requires a priori assumptions about the optimal number of components  in triplicate for combinations of q-value cutoffs between 0.005 and 0.1 and k between 10 and 120. We assessed the biological interpretability of the candidate solutions by enrichment for Gene Ontology Biological Process (GO:BP) terms. We found that optimizing only for maximum percentage of GO:BP-enriched GEPs yielded mostly large GEPs associated with very general biological processes. Under the assumption that a maximally interpretable set of GEPs should capture a wide range of biological processes and should favor discovery of minimally redundant GEPs, we then calculated two scores: one based on the breadth of GO:BP enrichments in a given ICA solution and the other on the unique assignments of GO terms to GEPs. We then rescaled these scores to a maximum of 1 and calculated a joint score by multiplying them together. For our final set of gene programs, we ran consensus ICA a final time with the k and q-value that maximized the average joint score across all three test replicates.To reduce bias and use of arbitrary cutoffs, we used a grid search approach to choose PRJNA475132) with fastp76 v0.20.0 and aligned to the custom reference using STAR26 v2.7.3 with chimeric junction detection turned on and “--chimScoreJunctionNonGTAG 0”. Other non-default parameters used are available via the linked github repository. We identified evidence of chimeric TE transcripts from the “Chimeric.out.junction” output file. We calculated normalized coverage for each strand using deeptools77 v3.3.1 “bamCoverage” command with “--smoothLength 150”.We trimmed poly-A selected RNA-seq , T4 DNA Polymerase (NEB M0203S), PolI Klenow fragment (NEB M0210S), and T4 Polynucleotide kinase (NEB M0201S). Adenylation was performed with 3′−5′ Klenow Fragment (NEB M0212L). Adaptors were ligated with NEB Quick Ligase for 10 min at 30 °C before two rounds of cleanup with homemade beads. NEBNext UltraII Q5 kit (NEB M0544) was used for PCR enrichment. A final round of cleanup with homemade beads was performed before quantification and sequencing.WGS library generation protocol was adapted from the Marshall-Lab DamID-seq protocol79 v3.2.0 with options “-q 20 -m 35.” We aligned trimmed reads with bwa-mem280 v2.0, removed duplicate reads with picard81 v2.22.1 with option “VALIDATION_STRINGENCY = LENIENT”, and filtered out multimappers with samtools82 v1.10.We trimmed reads using cutadapt83 v0.3.1 to calculate genome-wide read coverage in 100 bp bins for the autosomes and TE consensus sequences. We then calculated the median coverage value for each autosome  and each TE consensus sequence. We averaged the median coverage across autosomes to produce a single coverage value for autosomes. We then used the ratio of TE coverage to autosome coverage to generate estimated copy numbers for all TEs. These estimated copy numbers were then used to compare male and female copy numbers.To estimate TE copy numbers, we used mosdepth84 by finding mismatches with a base quality of at least 10 and at least 15 supporting male reads but lacking supporting female reads.We identified male-specific polymorphic sites with RsamtoolsWe used ~100 pairs of testes from 3 to 5-day-old mated w1118 males. The testes were dissected in 1X PBS and transferred into 200 µL RNAlater Solution. Tissue was pelleted by centrifuging at 5000 g for 1 min at 4 °C. Supernatant was removed and 300 µL 1× DNA/RNA Shield was added before homogenization with an electric pestle. Homogenized tissue was digested with Proteinase K at 55 °C for at least 30 min. RNA was purified with the Zymo Quick-RNA Plus Kit (R1057).Using up to 5 µg total RNA, ribosomal RNAs were removed using iTools rRNA depletion Kit from Galen Laboratory Supplies (dp-P020-000007) and Thermo Fisher MyOne Streptavidin C1 Dynabeads (#65001). RNA Clean and Concentrator-5 kit from Zymo Research (R1015) was used to purify rRNA-depleted RNA. Starting with 1 ng-100 ng purified rRNA-depleted RNA, Illumina libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760).76 v0.20.0. We used STAR26 v2.7.5 to align total RNA-seq reads to a bait reference composed of Flybase release 6.22 tRNA sequences and miscRNA sequences. We then aligned unmapped reads to our custom reference and provided STAR with a VCF file containing male-specific variants.For quantifying Y-linked expression for TEs, raw reads were trimmed with fastp26 v2.7.9 to align total RNA-seq reads to our custom genome sequence with parameters recommended by Arriba documentation. Arriba85 v2.1.0 was run with blacklist, uninteresting_contigs, read_through, intronic, long_gap, and intragenic_exonic filters disabled and “-U 32766”. Consensus TE sequence names were passed as viral contigs via the “-v” flag.For Y-gene fusion detection, raw reads were trimmed with fastp v0.20.0. We used STAR29, we found the number of unique polymorphic insertions on the X chromosome and on autosomes, excluding chromosome 4, across the Drosophila Genome Reference Panel for all TEs in our custom reference. For all TEs with at least 1 X-linked and 1 autosomal insertion among all DGRP lines, we calculated the ratio of X-linked or chromosome 4-linked insertions per megabase to autosomal insertions per megabase. To correct for possible differences in read mappability between chromosomes, we used the mappable lengths of each chromosome as reported by TIDAL-FLY29. In cases where there were differences in the TE family names between RepBase and TIDAL-FLY, we used NCBI BLAST88 Web v2.11.0 to confirm that the consensus sequences were identical or nearly identical and then treated the family names as synonyms.Using the TIDAL-Fly polymorphic TE insertion database40 as described above for the main reference sequence. For overall Y enrichment, we did not consider the terminal repeat portions of LTR retrotransposons, as we wanted to test the enrichment of potentially active copies. For Y-gene intron enrichment, we considered only Y-linked insertions but we did not remove LTRs, as solo LTRs could potentially contribute to detected transcription.We used RepeatMasker to identify insertions in a heterochromatin-enriched assemblyEAChm labeled with Quasar670 and against ACCORD2 and QUASIMODO2 labeled with CAL Fluor Red 610 were designed using Stellaris’ probe design tool available at www.biosearchtech.com/stellarisdesigner. Default parameters were used for EAChm probes. Probes against ACCORD2 and QUASIMODO2 were designed with masking parameter 2. To ensure specificity of the resulting probes, we used BLAST89 to align to consensus TE sequences used for masking and custom reference generation to ensure that all probes show complementarity to their intended target only. ACCORD2 and QUASIMODO2 probes were also blasted against Drosophila melanogaster REFSEQ sequences and any individual probes with more than 16 nucleotide matches to another sequence were removed from the final probe set. Probe sequences are available in Supplementary Data Custom Stellaris FISH probes recognizing Strain w1118 flies maintained at room temperature were mated for 4 h and offspring were grown at 25 °C until reaching the third instar. We dissected L3 males in sterile 1X PBS and fixed testis in 3.7% formaldehyde solution at room temperature for 45 min. Testis were washed twice with 1X PBS and submerged in 70% ethanol at 4 °C overnight. Hybridizations were carried out according to instructions available on the manufacturer website.90.Image slices were captured on a Carl Zeiss LSM880 AxioObserver with a C-Apochromat 40×/1.2 W Korr FCS M27 water immersion objective. 2D deconvolution was performed using ZEN Black software. Further contrast adjustments and image overlays were performed with Fiji91 “rmsk2bed” and TEs with copies within the range X:21631891-21790731 were identified as flamenco-regulated.RepeatMasker results (see above) for dm6.22 were parsed with bedopsPRJNA197267 and mapped with STAR26 v2.7.3a to the TE consensus sequence and r6.22 combined reference. DESeq292 was used to compare expression for each knockdown line to controls. TEs were considered to be regulated by the piRNA pathway in ovary if they were significantly upregulated after knockdown in at least 1 knockdown line .RNA-seq reads for piRNA pathway component knockdown lines were accessed from the NCBI SRA at Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the ‘master weaver of the genome’. Given its multiple connections, further characterization of CTCF’s emerging role in splicing regulation might extend its functional repertoire towards a ‘conductor of the splicing orchestra’. Since tNA motif . CombinaNA motif ,5 allowsNA motif . More thNA motif . UbiquitNA motif .CTCF has also been characterized as an RNA-binding protein and these interactions are essential for facilitating CTCF-mediated chromatin architecture . Such inThe complexity of CTCF interactions is further expanded by the ability of CTCF to homodimerize and mediate contacts with multiple distal intra- and inter-chromosomal target sites across the genome. Such interactions can facilitate genome folding into topologically associating domains (TADs) and chromatin loops, encompassing those interaction domains . This alGiven its unique role in higher-order chromatin architecture and the multiplicity and specificity of its binding sites, CTCF is able to regulate diverse molecular and epigenetic functions, summarized herein. CTCF is a unique regulator of gene expression, which can function as a transcriptional repressor and activator as well as a chromatin insulator, the latter occurring through interference of the contact between enhancers/silencers and promoters ,22. FurtCompelling evidence has linked CTCF to modulation of alternative splicing (AS) at both the individual transcript and transcriptome-wide level . AS is aCTCF’s involvement with AS, identified over the past decade, has expanded the list of mechanisms by which CTCF can regulate gene expression and transcriptomic complexity ; howeverIt is widely accepted that AS re-configures the transcriptome and proteome to facilitate certain biological processes. Perturbations to this highly calibrated process can cause diseases such as Hutchinson-Gilford Progeria syndrome, Duchenne muscular dystrophy, melanoma and breast cancer ,50. Thuset al. revealed the first direct link ‘roadblock model’ between CTCF and AS regulation involving RNAPII elongation and methylation of CTCF binding sites. Binding of CTCF to CD45 exon 5 impedes RNAPII elongation which allows the spliceosome to assemble at the weak upstream splice site and eventually leads to the inclusion of exon 5 in human Burkitt lymphoma B cells  elongation is fundamental to transcription and constitutive splicing processes, which require an optimal RNAPII elongation rate for producing canonical mature mRNA transcripts. This means that slowing or accelerating transcription elongation can both inhibit or enhance splice site recognition depending on additional factors. Alteration of the elongation rate influences inclusion of exons or introns particularly those having weak splice sites and short flanking introns . In a pi B cells  .The association of CTCF binding with RNAPII stalling at specific sites has been previously observed genome-wide in multiple cell lines , particu and p21 . Within  and p21 . Interes and p21 , which i  in different contexts  to specific amino acid residues on the target substrate ation (PARylation) activity ,61. PARyubstrate ,64. It mubstrate . While Cubstrate . A folloubstrate . PARP1, ubstrate ,69). Whiubstrate .Given its direct effect on PARP1 and its PARylation activity ,61, CTCFc-myc, and RNAPII termination at U2 snRNA gene via regulating the recruitment or activation of key factors, such as DRB-sensitivity-inducing factor (DSIF) and negative elongation factor (NELF)  . Moreover (NELF) . The worocessing . WhetherIntragenic CTCF binding sites, particularly those proximal to splice junctions, influence pre-mRNA splicing decisions, and thus mediate alternative exon or intron inclusion ,40–42,44Pcdh gene family, which encode multiple isoforms responsible for neuronal cell surface diversity  levels  levels . Likewis) levels ). Knowin) levels ,40,73, iNucleosome positioning is known to modulate RNAPII kinetics by acting as a physical barrier hindering RNAPII elongation and thus promoting the recognition of exon-intron boundaries by the splicing machinery . NucleosInterestingly, several studies have reported an enrichment of CTCF sites around nucleosomes genome-wide, indicating that CTCF acts as an anchor for positioning nucleosomes in a symmetrical distribution ,85–87. YConstitutive splicing depends on many factors including the guanine and cytosine (GC) content, which is higher in exons compared to introns. DNA methylation contributes to the definition of exon-intron splice sites depending on the enrichment of CpG dinucleotides within the spliced exons/introns ,92. It hCacna1b transcription, where CTCF binding to hypomethylated exon 37a (but not 37b) promotes its recognition and inclusion in neurons , in a seemingly RNAPII elongation-independent manner , and generates this truncated BDNF2a isoform. Interestingly, TET1 binding at MeCP2 sites was also essential for BDNF splicing, which is inhibited by knockdown of MeCP2, YB1 or TET1 , and the Cancer Cell Line Encyclopedia , which are useful for comprehensive analysis of AS landscapes in various normal and malignant cells. Given frequent allelic variations and somatic mutations in CTCF . Th in CTCF , it woul in CTCF , a new rStructural changes in the genome arising from genetic variation or introduced experimentally via deletion or inversion of CTCF sites using genome editing can have pathological consequences. Disrupting the highly conserved architecture of TADs has been shown to have a dramatic impact on development and normal spatio-temporal control of gene expression . While tTo date, the role of CTCF in AS has been mostly elucidated using computational approaches, while experimental validation remains challenging especially at a genome-wide level. This is further complicated by the fact that CTCF is essential for somatic cell viability , genome


We have recently revealed the existence of a cell cycle-regulated chromatin segregase, Yta7 , involved in chromosome replication. Phosphorylation of Yta7 by S-CDK (S-phase Cyclin-Dependent Kinase) regulates its function. These findings link the cell cycle to chromatin biology and suggest how chromatin-modifying enzymes become S phase-specific. This is because of the inherent stability of nucleosomes. To access DNA packed in chromatin, cells require the help of chromatin-modulating factors like chromatin remodelers or histone chaperones.Genome duplication is one of the most fundamental processes a cell undertakes. Eukaryotic genomes consist of DNA wrapped around histone octamers to form nucleosomes, the basic unit of chromatin.in vitro.2 However, in vivo, these chromatin factors also play important roles during other processes, most prominently gene transcription. Transcription takes place throughout the cell cycle, and it remains unclear how these chromatin factors were made “S phase-specific” to support chromosome replication.In contrast to gene transcription, chromosome replication is special in the way that it takes place exclusively during the S-phase of the cell cycle. It has been shown that ATP-dependent chromatin remodelers such as INO80 complex (INOsitol requiring 80) or ISW1a (Imitation Switch subfamily), the histone chaperone FACT (FAcilitates Chromatin Transcription)/Nhp6  along with the histone acetyl transferases SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (NUcleosome Acetyltransferase of H4) are necessary for chromatin replication 3 This makes an important step forward toward understaning how chromatin factors are regulated at specific cell cycle stages. Yta7 was previously described as a chromatin boundary element,4 and it was shown that it influences nucleosome density and histone gene transcription.6 The molecular underlying molecular mechanism, however, remained elusive. A idea at the time was that Yta7´s boundary function prevents spreading of repressive chromatin structure into histone gene coding sequences by keeping histone chaperone Rtt106 (Regulator of Ty1 Transposition protein 106) and the chromatin remodeler RSC (Remodeling the Structure of Chromatin) in place.5 Another exciting model was that Yta7 actively influences chromatin structure and thus influencing histone gene transcription.6 However, there were no biochemical data supporting either hypothesis.In our study, we revealed that Yta7  is a novel type of chromatin factor, which activity to promote chromosome replication is regulated by phosphorylation by S-CDK.7 Yta7 assembles into ~1 mega Dalton hexameric complexes that can efficiently bind acetylated chromatin. Interestingly, Yta7 also bears a bromo-domain; however, if this domain is important for the association with acetylated histones is unknown. Interestingly, S-CDK-dependent phosphorylation in close proximity to the AAA+-ATPase domain stimulates ATP hydrolysis and activates the chromatin segegase function of Yta7. As a result, Yta7 disrupts chromatin by disassembling nucleosomes, which facilitates progression of the replisome through a chromatin template, thus preventing replicative stress or genomic instability.3We now showed that Yta7 is a chromatin segregase that uses the energy of ATP hydrolysis to disrupt chromatin structure. Yta7 is very disting from classical chromatin remodelers of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family. It bears two AAA+-ATPase modules, where one is highly conserved among species.9 ATAD2 levels in normal proliferating cells are tightly controlled by mitogenic factors via the retinoblastoma (RB) - E2 transcription factor (E2F) pathway. During tumorigenesis, ATAD2 is highly expressed and disturbes the RB-E2F pathway, which contributes to tumor development.9 ATAD2 has been reported to function as a transcriptional co-regulator for several oncogenic transcriptional factors such as estrogen receptor or the androgen receptor and the transcription factors E2F and MYC (Master Regulator of Cell Cycle Entry and Proliferative Metabolism).9 It has been suggested that ATAD2 might link ocogenic and proliferation pathways and that this drives to the development of aggressive tumors.Yta7 has a human homologue ATAD2 (ATPase family AAA+ domain-containing protein 2), which is an emerging oncogene and has been associated with many cancers of different origins.10 Interestingly and in line with our findings, ATAD2 has been shown to express in S phase where it then localizes to replication sites. ATAD2 multimerizes through its AAA+-ATPase domain and interacts with acetylated marks on newly synthesis histones to aid heterochromatin compaction. Also, ATAD2 is co-expressed with genes involved in DNA replication in various cancer types,8 suggesting that, like Yta7, it might play a role in chromosome replication in multicellular eukaryotes as well.Activated oncogenes influence replication by altering replication timing and progression and causes replicative stress but how they accomplish this remains poorly understood.Because of our discovery and due to the high level of conservation, we propose that ATAD2 might function similarly to Yta7 to influence chromatin structure directly, which is important for faithful and timely chromosome replication.


In human cells, each rDNA unit consists of the ~13 kb long ribosomal part and ~30 kb long intergenic spacer (IGS). The ribosomal part, transcribed by RNA polymerase I (pol I), includes genes coding for 18S, 5.8S, and 28S RNAs of the ribosomal particles, as well as their four transcribed spacers. Being highly repetitive, intensively transcribed, and abundantly methylated, rDNA is a very fragile site of the genome, with high risk of instability leading to cancer. Multiple small mutations, considerable expansion or contraction of the rDNA locus, and abnormally enhanced pol I transcription are usual symptoms of transformation. Recently it was found that both IGS and the ribosomal part of the locus contain many functional/potentially functional regions producing non-coding RNAs, which participate in the pol I activity regulation, stress reactions, and development of the malignant phenotype. Thus, there are solid reasons to believe that rDNA locus plays crucial role in carcinogenesis. In this review we discuss the data concerning the human rDNA and its closely associated factors as both targets and drivers of the pathways essential for carcinogenesis. We also examine whether variability in the structure of the locus may be blamed for the malignant transformation. Additionally, we consider the prospects of therapy focused on the activity of rDNA. In human cells, ribosomal DNA (rDNA) is arranged in ten clusters of multiple tandem units situated on the short arms of five chromosomes  ,7,8. TheIn this review we focus on the data concerning the human rDNA and its closely associated factors as both target and regulator of the pathways essential for carcinogenesis. We also examine whether some variability in the structure of the locus, may trigger the malignant transformation. Additionally, we consider the therapy targeting the activity of rDNA.Nucleolus is a highly dynamic structure and quickly responds to changing proliferative or environmental cues, including alterations of the major tumor suppressor pathways, which makes this organelle a mirror of the malignant transformation ,9. MorphTherefore, disorganization of either ribosomal or extra-ribosomal functions of nucleoli may be a key permissive step for malignant transformation .Tumors are characterized by high levels of ribosome production due to their reliance on cell proliferation and increased protein synthesis. Therefore, transcription of rDNA by pol I, which is a crucial step in ribosome biogenesis, plays a central role in cancer ,21,22,23The transcription factor Myc (product of the c-Myc oncogene) is one of the most frequently activated oncoproteins, overexpressed in ~50% of all cancers . Myc is The two major tumor suppressor proteins, p53 and retinoblastoma protein (pRb), control proliferation in mammalian cells. In human cancers, the function of the suppressors is frequently altered, so that they may be involved in tumorigenesis. These changes lead not only to the loss of control on cell proliferation, but also to up-regulation of rRNA transcriptional activity . ProteinBasonuclin is a zinc-finger protein usually found in epithelial and germ cells, but its expression is very high in basal cell carcinoma of skin. Normally this protein binds to rDNA promoter and takes part in the initiation of the pol I transcription. Apparently, dysregulation of basonuclin function may contribute to carcinogenesis .DNA methylation is one of the major factors which determine the gene expression and progression of human diseases is often accompanied by changing the rDNA methylation pattern ,41,42. TThe distribution of the CH3 groups along the DNA strand presents a special problem. The methylation of promoter is usually believed to be a repressive epigenetic mark, although in senescent cells the rRNA coding regions are highly methylated as well ,48. StraThus, methylation of rDNA locus, especially its promoter, is one of the key factors of carcinogenesis, but the phenomenon is complicated.Being highly repetitive, heavily transcribed, and methylated, rDNA is a very fragile spot of the human genome, with high risk of instability leading to cancer ,56 Figu. RepetitRepair of the damaged rDNA repeats may engage sister chromatids, homologous as well as non-homologues chromosomes, or even repeats in cis position as repair templates. However, the restoration of damaged sites may be accompanied by interchromatid crossovers, translocations, generation of acentric/dicentric chromosomes, repeat expansions and contractions, and/or extra-chromosomal circles ,62. ThusNormally, the unfavorable changes in the locus are suppressed by the packaging of repetitive sequences as constitutive heterochromatin, which is typically associated with histone H3 lysine 9 methylation, the heterochromatin protein 1a (HP1a) binding, histone hypoacetylation, and expansive DNA methylation. Such chromatin structure potentiates pairing, regulates repair, and inhibits recombination ,65. TherSmall-scale DNA polymorphism represented by single nucleotide variants (SNV), short insertions and deletions, which regularly occur as errors of replication, seems to be another important factor of carcinogenesis. Correlation between malignant transformation and high frequency of the small-scale variations have been well demonstrated ,68. In eBut, although rDNA instability seems to be a common feature of cancer and other severe health problems ,69, it iProbably the best studied diseases connected with disruption of the ribosome biogenesis are ribosomopathies. These rare diseases increase the risk of carcinogenesis, but they all seem to be consequences of mutation in ribosome protein and ribosome-related processor genes, not in the rDNA locus itself ,71.It has been demonstrated that malignant transformation correlates with instability of rDNA locus; but so far this instability has not been proved to be a primary cause of carcinogenesis.Even in normal state, the number of copies in the rDNA locus varies among individuals, individual cells, and the rDNA bearing chromosomes within one cell. But this variability increases usually by 20–80% in pathology, especially when the cells respond to the DNA damage ,72,73,74The increased instability of rDNA copy number may have multiple effects on the nucleolar and nuclear functions including telomere maintenance, transposable element silencing, and satellite DNA stability, which are important factors of tumor suppression ,82,83. BThe distribution of the functional regions in the locus are shown in The RNAs of 21–23 nt long and various structure, involved in gene expression and other processes, are known as miRNAs. It is believed that all human genes may fall under control of these short molecules . Even smThis group of miRNA-like molecules, varying in size and structure, derive from the precursor and mature rRNAs, as well as IGS, in a poorly understood manner. Some srRNAs may emerge as mere fragments of random degradation, which is a necessary phase of rRNA metabolism . But manQDE-2-interacting small RNAs, or qiRNAs are interference RNAs induced by DNA damage. QiRNAs originate mostly from the rDNA locus and participate in DNA damage response in the filamentous fungus Neurospora crassa. It has been reported, that in human cells qiRNA may be affected in diabetes, Perlman syndrome, Wilm’s tumor development, early embryogenesis, and stem cell proliferation ,102.gRNA typically binds to DNA and anti-sense RNA sequences by complementary base pairing. Binding of gRNA to RNAs results in their post-transcriptional modifications, such as ribomethylation and introducing pseudouridine. Thus, modified RNA and DNA sequences may be targets of deletion, insertion and other alterations. Some of human gRNAs originate from rDNA locus .Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA, mostly involved in the epigenetic and post-transcriptional silencing of transposable elements and other repeat-derived transcripts n simple repeats and situated at ~16 kb, ~22 kb and ~28 kb are called rIGS16, rIGS22, and rIGS28, respectively. These regions produce ~300 nt long RNAs, which in response to stress, such as acidosis or heat shock, detain within the nucleoli, and temporarily inactivate certain enzymes, including components of the DNA polymerase complex. The process is accordingly named “nucleolar detention” [Three regions of IGS enriched in [TC]tention” . Since tPyrimidine-rich non-coding transcript (PNCTR) is another kind of lncRNA transcribed from the ~28 kb IGS region. This pol I transcript is >10 kb in length and its pyrimidine-rich sequence harbors multiple putative binding sites of polypyrimidine tract-binding protein, that is associated with primary transcripts and regulates their processing. Remarkably the PNCTR is much more abundant than the detaining lncRNAs and shows higher levels in cancer cells compared to normal cells .p < 0.01) compared to dominant cancer genes (oncogenes) and the entire set of genes in the human genome [The human genome contains about 10% Alu elements, the mutable DNA sequences of ~300 nt in length belonging to the class of retrotransposons. These elements are particularly abundant in rDNA, where they are for the most part incomplete and unable to migrate . Alu repn genome . Recentln genome . On the n genome ,130. Aftn genome . Thus, AHypothetically, many other regions of rDNA locus may be functional even without producing ncRNAs . Functional regions will be very likely found among the numerous repetitive sequences within IGS . These sIt has been long known that a number of conventional chemotherapeutic agents impair ribosome biogenesis and/or nucleolar size, number and morphology. A recent screen of common chemotherapeutic drugs demonstrated that out of 36 agents tested, 21 were found to affect rDNA transcription, early rRNA processing (measured by the occurrence of the 32S rRNA intermediate) and late rRNA processing (measured by the occurrence of the mature 28S or 18S rRNAs) . The plaRecent studies show that rDNA plays a crucial role in carcinogenesis. The locus has been described as a target for the essential pathways of malignant transformation. In this respect, the data on the nucleolar proteins, gain/loss of the copy numbers, abnormal transcription enhancement, and promoter methylation/demethylation seem to be conclusive. Moreover, it is believed that alterations in the rDNA structure and functions may be actual drivers of the malignant phenotype ,26. Some


Fungal secondary metabolites are small molecules that exhibit diverse biological activities exploited in medicine, industry and agriculture. Their biosynthesis is governed by co-expressed genes that often co-localize in gene clusters. Most of these secondary metabolite gene clusters are inactive under laboratory conditions, which is due to a tight transcriptional regulation. Modifications of chromatin, the complex of DNA and histone proteins influencing DNA accessibility, play an important role in this regulation. However, tinkering with well-characterised chemical and genetic modifications that affect chromatin alters the expression of only few biosynthetic gene clusters, and thus the regulation of the vast majority of biosynthetic pathways remains enigmatic. In the past, attempts to activate silent gene clusters in fungi mainly focused on histone acetylation and methylation, while in other eukaryotes many other post-translational modifications are involved in transcription regulation. Thus, how chromatin regulates the expression of gene clusters remains a largely unexplored research field. In this review, we argue that focusing on only few well-characterised chromatin modifications is significantly hampering our understanding of the chromatin-based regulation of biosynthetic gene clusters. Research on underexplored chromatin modifications and on the interplay between different modifications is timely to fully explore the largely untapped reservoir of fungal secondary metabolites. This review focuses on the chromatin-based regulation of fungal secondary metabolite biosynthesis and argues that the incomplete picture of chromatin modifications in fungi significantly hampers the activation and exploitation of fungal secondary metabolites in medicine, industry and agriculture. Penicillium chrysogenum through its anti-bacterial activity. This compound was subsequently developed into the first broad-spectrum antibiotic that provided the first effective medication against bacterial infections and started the antibiotic era in modern medicine . SMs exhibit a wide range of biological activities, some of which had and have significant impact on human societies. For instance, penicillin was accidentally discovered in 1928 by Alexander Fleming from the mould ns Balba . Other fStreptomyces  successfully induced the production of novel compounds  proteins – the chromatin. The basic ‘unit’ of chromatin is the nucleosome, which is formed by DNA wrapped around a histone octamer of two copies of each of the four core histone proteins   has been observed in most animals, plants and fungi   et al. et al. et al. et al. et al. et al. et al. et al. The removal or addition of specific histone PTM is catalysed by writer and eraser proteins Table . For exaN. crassa, H3K9me3 co-localizes with DNA methylation and forms constitutive heterochromatin at telomeres, centromeres and transposon-rich regions throughout the genome , containing the histone methyltransferase (KMT1) Dim-5 (a homolog of the Schizosaccharomyces pombe Clr4), which, in turn, directs cytosine methylation by recruitment of heterochromatin protein 1 (HP1) and the DNA methyltransferase Dim-2 , F. graminearum (kmt6), or F. fujikuroi (kmt6) led to the loss of H3K27me3 and to the activation of hundreds of genes, many of which are involved in SM biosynthesis  and lolitrems (ltm), two SMs with bioprotective activity against herbivores (Chujo and Scott EfclrD (kmt1) or EfezhB (kmt6) methyltransferases led to a reduction of H3K9me3 and H3K27me3, respectively, and to the de-repression of eas and ltm gene expression in vitro , which encodes another COMPASS component, resulted in strong reduction of H3K4me2/3 and changes in SM profiles, for instance the activation of the monodictyphenone and F9775A/F9775B SM gene clusters in A. nidulans , which is the catalytic subunit of the COMPASS complex  between different modifications  display de-regulation of many SM gene clusters that are localized within sub-telomeric regions  H3K9me3-rich regions  facilitated the genome-wide exploration of major chromatin modifications, mainly histone acetylation and methylation  or hp1 affects chromatin architecture only mildly, while deletion of set-7 (loss of H3K27me2/3) or the importin alpha dim-3  decreased interactions between constitutive heterochromatin compartments  methods allows to investigate the 3D architecture of genomes. Hi-C sequencing revealing contacts between genomic loci at the whole-genome scale in an untargeted fashion . Fungal symbionts often produce specific chemical compounds to establish and support symbioses. For instance, the ectomycorrhizal basidiomycete fungus Laccaria bicolor produces sesquiterpenes to promote lateral root formation in plant hosts (Ditengou et al. Interactions between species occur in diverse ecological niches. In particular, fungi compete with other microorganisms that live in the same niche and thus produce antimicrobial compounds, both peptides and SMs, to colonise their niche. For instance, the co-cultivation of in vitro conditions (e.g. Connolly et al. et al. in planta contact maps were not studied in E. festucae (Winter et al. et al. Unfortunately, studying histone and DNA modifications during host interaction suffers from technical challenges. In early stages of host colonisation, when SM gene clusters are typically expressed, fungal biomass is low compared with abundant host biomass. Unsurprisingly, chromatin dynamics during host interactions thus far focused on few specific SM gene clusters using targeted approaches such as ChIP-PCR assays (e.g. Chujo and Scott The discovery of chromatin as a central regulator of fungal SM production significantly impacted on our understanding of the complex transcriptional regulation of biosynthetic gene clusters. The rapid progress in identifying the genetic determinants underlying this chromatin-based regulation provided new tools to activate silent gene clusters. Thus far, most efforts have focused on two well-known chromatin modifications, histone methylation and histone acetylation. However, most SM gene clusters remain untouched by any genetic or chemical modification of chromatin, and thus we still lack a comprehensive understanding on how chromatin modifications regulate SM gene clusters. Based on examples from other eukaryotes and the availability of novel technologies that allow to comprehensively study the composition and organisation of chromatin, we are now entering a new decade during which several outstanding questions can be answered. What is the contribution of other chromatin modifications to the regulation of SM gene clusters? How do chromatin modifications interact with each other to provide a tight and subtle regulation of SM gene clusters? How are chromatin modifications orchestrated in the different genomic sub-compartments, and what is the chromatin dynamic during interactions with other organisms? Answering these questions is important to advance our basic knowledge on chromatin-based regulation, but it will also provide the needed tools to successfully activate the wide diversity of fungal SM gene clusters. Exploitation of the fungal kingdom to the discovery and development of novel bioactive compounds would then enter a completely new era.


Insulin expression, the molecular mechanisms that dictate the function of PDX1 in response to glucose are still elusive. The transcriptional activation functions of PDX1 are regulated, in part, by its two intrinsically disordered regions, which pose a barrier to its structural and biophysical characterization. Indeed, many studies of PDX1 interactions, clinical mutations, and posttranslational modifications lack molecular level detail. Emerging methods for the quantitative study of intrinsically disordered regions and refined models for transactivation now enable us to validate and interrogate the biochemical and biophysical features of PDX1 that dictate its function. The goal of this review is to summarize existing PDX1 studies and, further, to generate a comprehensive resource for future studies of transcriptional control via PDX1.The pancreatic and duodenal homeobox 1 (PDX1) is a central regulator of glucose-dependent transcription of insulin in pancreatic β cells. PDX1 transcription factor activity is integral to the development and sustained health of the pancreas; accordingly, deciphering the complex network of cellular cues that lead to PDX1 activation or inactivation is an important step toward understanding the etiopathologies of pancreatic diseases and the development of novel therapeutics. Despite nearly 3 decades of research into PDX1 control of  PDX1 gene is located at chromosome 13q12.1 in Homo sapiens and at 5qG3 in Mus musculus (mouse); unless otherwise specified; discussion herein will focus on the H. sapiens protein PDX1. In addition to its role in developmentally mature organisms, PDX1 is of particular importance in the early differentiation of gut endoderm cells into pancreatic progenitors and, subsequently, the formation of insulin-producing cell mass  . Increased insulin in response to glucose stimulus is primarily a consequence of translation of proinsulin mRNA, thus replenishing the cell’s insulin stores . To gain insight into how PDX1 orchestrates INS transcription, we must first interrogate the many reported PDX1 interactions and modifications associated with its activating function.While not the specific focus of this review, glucose sensing and insulin secretion by the β cell are inherently related to PDX1 action; accordingly, we provide a high level summary of these β cell processes. Briefly, Glut1/3 mediates glucose transport into the β cell, after which the glucose is processed by glycolysis in the cytoplasm and through the Krebs cycle in the mitochondria. The resulting increase of cytoplasmic ATP concentration blocks potassium efflux, leading to a change in membrane polarization that ultimately stimulates exocytosis of insulin granules , 27 FigB, top. In stores . In contINS mRNA , 31, 32.INS mRNA , 34 , transcription factors tend to contain intrinsically disordered transactivation domains (TADs) that support interaction with posttranslational modification (PTM) enzymes, chromatin remodelers, transcriptional coactivators or repressor proteins, and other transcription factors , 39, 40.Many IDR interactions are tailored by their covalent modification in or near a binding surface; such modification may be coupled to conformational consequences, but this is not universally required , 47. DueInsulin, iapp, and glucokinase gene promoter elements permits differential PDX1 binding to the various promoter sites  . Amino and turns . Althougnd turns .P52945). Proline, glycine, and charged and polar amino acids are generally considered ‘disorder promoting’; in contrast, hydrophobic residues tend to be associated with folded protein structures  is also intrinsically disordered; in contrast to many IDPs and IDRs, which have some degree of transient or low population secondary structure, solution-state studies of PDX1-C by NMR spectroscopy and small angle X-ray scattering support the disorder prediction that this region is devoid of persistent structure  62). PD. PD62). protein–protein interactions section), PDX1-C has only one annotated binding partner, speckle-type POZ protein (SPOP) , which facilitates PDX1 proteasomal degradation  protein, which is a well-known tumor and cell cycle suppressor, interacts with a specific structural motif within PDX1-HD. The PDX1-RB interaction was identified by overexpression in and coimmunoprecipitation (co-IP), although the experiments were performed in a non-β cell line. Hyperphosphorylated RB (pRB) associates with the Pdx1 RB interaction motif found in Helix 2 of the HD fold see  and doesThere is little available information describing specific molecular mechanisms for PDX1 association with its various interaction partners. Thus, it is imperative that efforts be directed to understanding the atomistic determinants of binding and the cellular signals (such as glucose stimulus) that promote or preclude interactions with PDX1. A major barrier to understanding the regulatory roles of PDX1-interacting factors is the absence of high-resolution structural information describing the binding interfaces. The X-ray crystal structure of the PDX1 HD in complex with DNA led to a model for induced fit for PDX1 recognition of and binding to DNA elements . CrystalINS transcription  associate directly or indirectly with PDX1; some such factors bind nearby promoter or enhancer elements toward activation of cription , 73.versus Brm occurs at genes (including INS) that are activated by PDX1  alters nucleosome positioning by sliding or ejecting nucleosomes to facilitate transcriptional activation or repression . The int by PDX1 , 75. In in vitro and in vivo with the proline-rich domain of an Epstein-Barr viral protein, EBNA2 . In contrast to the established role of Set7 as a monomethyltransferase, dimethylated H3K4 was the primary species identified at active INS promoters, which implies the involvement of a yet unidentified secondary methyltransferase enzyme  , 88. LikA). Informed by structural studies of nonhistone proteins in complex with p300, we hypothesize that PDX1 binding occurs in a similar fashion. In the case of E47, two small helical domains of p300, TAZ1, and TAZ2 compete for binding to an acidic IDR of E47  and β2 ; the E47/β2 heterodimer binds E-box motifs near to Pdx1-bound A-box DNA motifs (via an HD-containing region (residues 138–213) based on pull-down experiments with in vitro–translated PDX1 constructs . Although Bridge-1 does not appear to have transcription factor activity, transcription reporter assays and pull-down studies showed that it associates with both E47 (but not β2) and PDX1 toward enhanced tivation , 101. Torphology . A proterphology . Taken tThe high mobility group A1 protein  is an architectural factor that binds to DNA and other transcription factors but does not itself have intrinsic transcription factor activity. Instead, HMGA1 binds AT-rich minor grooves and modulates the DNA conformation toward additional transcription factor recruitment and binding , 105. HMHMGA1 impairs endocrine pancreas function and is associated with the development of insulin resistance and diabetes. Indeed, HMGA1-deficient patients exhibited impaired insulin secretion and reduced binding to the INS promoter by PDX1 . In contrast to the several factors that are involved in INS activation via PDX1, there are fewer known proteins that serve as corepressors.In glucose-starved conditions, expression of ter site , 112. AsINS promoter decrease alongside a concomitant increase in occupancy by both HDAC-1 and HDAC-2 , which generally are contained within larger complexes, such as NuRD, remove histone acetylation marks at repressed genes . Indeed,d HDAC-2 .Perhaps expectedly, given the phosphorylation-dependent recruitment of an acetyltransferase, HDAC-1/2 binding to PDX1 relies on TAD dephosphorylation. Although the phosphatase(s) responsible for PDX1 dephosphorylation have not been experimentally validated, treatment of cells with an inhibitor of the phosphatases PP1 and PP2A disrupted HDAC-1/2 interaction with PDX1. Notably, however, deletion of the TAD region leads to constitutive—irrespective of glucose—interaction of PDX1 with HDAC-1/2 . In otheThe Swi/Snf complex is also recruited to PDX1 under low glucose conditions but now containing the Brm ATPase instead of Brg1 , 76. BrmINS, Sox6, has been identified and characterized .To our knowledge, there are no reports of crosstalk between the N- and C-terminal repressive mechanisms that downregulate in bound . From thin bound B.https://www.cbioportal.org/) reports PDX1 mutations identified in patients with other cancers, including lung, bladder, uterine, liver, and colorectal cancers , is linked to the onset of diabetes , 137. TyPDX1 are classified as MODY4 .As established previously, genomic or posttranslational dysregulation of PDX1 is linked to cancer and diabetic phenotypes. In addition to the contribution of PDX1 to polygenic diabetes (T2D), PDX1 is also implicated in pancreatic agenesis, neonatal diabetes, and MODY phenotypes , 148. Inpatients A.Figure PDX1 mutation usually depends on the mutation type and zygosity. For instance, a frameshift mutation leading to PDX1 truncation N-terminal to the DBD is a causative factor in mild MODY4 (heterozygous) and neonatal diabetes resulting from pancreatic agenesis (homozygous) (via decreased DNA binding (INS by PDX1 as a consequence of attenuated DNA binding.Phenotypic severity associated with ozygous) , 151. Si binding , 157. InA). PDX1 harboring a C18R or P33T mutation in the N-terminal TAD leads to impaired β cell differentiation from pancreatic progenitor cells and decreased insulin, glucagon, and somatostatin activation in mature cells  of the PDX1 IDRs A. PDX1 hre cells , 135. PDre cells , 158. A re cells , 160 . Another C-terminal substitution, P239Q, was identified in a patient with MODY3 and was linked to a more severe diabetic phenotype compared with the MODY3 mutation alone .PDX1 with proline insertion at position 243 or point mutation resulting in E224K both had decreased transcription activation ability compared with the WT, but neither mutation was deemed causative , 161. NoAs emphasized by the disease consequences of dysregulation of PDX1 or the genes under its control, appropriate regulation of PDX1 function is integral to organism health. Accordingly, intrinsically disordered transcription factors, such as PDX1, are subject to many layers of regulatory control.INS promoter, (ii) recruitment of chromatin remodelers and additional transcription factors, and ultimately, (iii) activation of INS expression. For the latter to occur, however, requirement is that PDX1 tune its intermolecular interactions for gene activation. While it is generally recognized that PDX1 transactivation potential is regulated through glucose-linked kinase pathways, the specific PTMs that direct PDX1 activation versus inhibition are not well established  PDX1 association with the ablished , 163. Thablished . Table 3PTMs are integral to protein function for IDPs and folded proteins alike; however, compared to their folded counterparts, IDPs are generally overrepresented as targets of such modifications , 167. InC). Threonine 11 (T11) is highly conserved in mammals only, despite the modest conservation of the flanking amino acids in other species.Phosphorylation events in the PDX1 TAD are primarily implicated in the control of PDX1 transcription factor activity. However, evidence of upregulation or downregulation through specific phosphorylation events is contradictory in the literature. Additionally, although most PTMs in PDX1-N are activating in nature, phosphorylation of PDX1 threonine 11 does not directly affect transactivation potential but appears to mark PDX1 for proteasomal degradation in response to DNA damage . AlignmeFig. 4Serine residues 61 and 66 (S61 and S66) lie within the TAD and their phosphorylation by ERK1/2 in response to glucose stimulus enhances PDX1 transactivation . A separINS. Given these results, it is possible that activation of PI3K by glucose may drive activation of downstream kinases in a pathway that is independent of p38/SAPK2 and ERK1/2 . Notably, the residues surrounding these phosphorylation sites are not directly conserved but maintain remarkable chemical similarity across species. This pattern is consistent with a model for a careful balance of acidic and hydrophobic residues within TADs, in which conservation of amino acid identity is less important than maintaining the more general charge and hydrophobicity features of a TAD sequence  responsible for the dephosphorylation of either PDX1 IDR.Although we know that glucose-dependent phosphorylation events in the PDX1 TAD tailor recruitment of certain factors as a manner of in vitro measurements of binding affinity to SPOP revealed that pT230 and pS231 modifications preclude SPOP binding and, thus, stabilize PDX1 (C). In addition, conservation of these phosphorylation sites and the nearby residues is also likely important for regulation of PDX1 stability via the SPOP interaction motif that encompasses T230 and S231.The PDX1 C-terminal IDR contains four putative phosphorylation sites; the phosphorylatable residues all lie within motifs recognized by the only known PDX1-C-interacting protein, SPOP. However, different studies report different consequences for phosphorylation: threonine 230 (T230) and serine 231 (S231) were initially proposed to be targets of casein kinase 2 (CK2) phosphorylation toward PDX1 turnover and subsequent lowered transcriptional activity , 183. Stize PDX1 . In eithize PDX1 C. In addC). Although the proposed kinase differs between studies, phosphorylation of S268, S272, or S268/S272 appears to drive proteasomal degradation of PDX1  motif. Sequence alignment shows high conservation of both sites in mammals but the absence altogether of this motif in some species of bird and fish C. Althou of PDX1 , 123. On of PDX1 . This fiInterestingly, another study suggested the HD-interacting protein kinase 2 (HIPK2) as the enzymatic source of the low glucose phosphorylation of S268 but reported localization of phosphorylated PDX1 to the nuclear periphery as the repressive mechanism, which may yield degradation-independent suppression of PDX1 activity . The mecC). Importantly, however, it is not clear whether this conservation is suggestive of a similarly conserved phosphorylation event or a consequence of high sequence homology across HD folds (Phosphorylation within the HD at position T151 is reported to control nuclear import of PDX1. In experiments using phosphomimetic (T151D) or phosphodeficient (T151A) mutants of PDX1, PDX1 harboring T151D was predominantly localized to the cytosol regardless of glucose stimulus. In this model, PDX1 phosphorylated by Per-Arnt-Sim kinase (PASK) at T151 (pT151) may be excluded or exported from the nucleus . PutativFig. 4HD folds .in vitro assays. Of the two, methylation of Lys131 conferred increased transcriptional activity in luciferase reporter assays; however, the mechanism by which this modification impacts PDX1 transactivation potential is not clear  protein associated with a phosphorylation event was proposed, in part, based on anomalous SDS-PAGE migration of PDX1 isolated from β-like cells compared with PDX1 expressed in Escherichia coli. The lysine residue that is modified by SUMOylation was not reported, but knockdown of SUMO-1 reduced PDX1 transcriptional activity in reporter assays and decreased PDX1 half-life in cells (O-linked N-acetylglucosamine (O-GlcNAc) and that this modification was associated with high-glucose conditions and insulin secretion. Modification of Ser/Thr by glycosylation may compete with phosphorylation events; however, the sites of modification by O-GlcNAc groups were not reported  that are modified to this end are yet unknown , 118. GiFig. 4in cells . Anotherreported .i.e., insulin secretion), studies of PDX1 PTMs must not be limited to identification of modifications. Instead, efforts should focus on describing the enzymes responsible for installation and removal of modifications and the molecular level consequences of PTMs in the different domains of PDX1.In order to establish rigorous connections between glucose conditions, PDX1 PTMs, and metabolic outcomes  or T2D.Of particular important focus toward understanding the signals that lead to PDX1 transcriptional activation is the study of the enzymes responsible for the placement and removal of PDX1 PTMs and the spatial and temporal specificity with which they do so. As a result, we may link glucose condition-specific PTM events with enhanced or impaired interactions with the modified regions of PDX1. By evaluating the existing literature surrounding PDX1 modifications and the PDX1 interactome, we may begin to make hypotheses about the possible effects of some PTMs and, thus, how PTMs exert control over PDX1 function.B). Separately, the proposed methylation events in the PDX1 N-terminal IDR could serve a similar role by blocking ubiquitination with an existing PTM.Testing one such hypothesis may, for example, reveal a mechanism for the stabilizing effect of pRB binding to PDX1. The lysine residue(s) modified within the PDX1 ubiquitin-mediated degradative pathway is unknown; however, pRB binding to the PDX1 HD see  reportedFig. 4in vitro approaches to complement in vivo studies of PDX1 phosphorylation. NMR is a tractable strategy to study possible structural consequences of PDX1 PTMs; we and others have leveraged NMR strategies toward kinetic and structural characterization of PTMs in disordered protein systems (The several PTMs, with emphasis on phosphorylation, are high-value targets of study toward understanding the glucose-linked signals that lead to PDX1 upregulation or downregulation. However, as evidenced by contradictory enzyme assignments and ambiguous consequences on PDX1 functions, we must explore  systems , 193.ThrDecades of work toward clinical descriptions of PDX1 function enables us now to connect healthy and disease phenotypes to specific molecular mechanisms of transcriptional control. In addition, study of the PDX1 IDRs in this context may be generally applicable to other IDR-containing transcription factors and shed new light on the many layers of regulation of proteins involved in gene expression processes.The authors declare that they have no conflicts of interest with the contents of this article.


Drosophila embryo has provided a powerful setting to directly observe the establishment and elaboration of chromatin features and experimentally test the causality of transcriptional events that are shared among many metazoans. The large embryo is tractable by live imaging, and a variety of well-developed tools allow the manipulation of factors during early development. The early embryo develops as a syncytium with rapid nuclear divisions and no zygotic transcription, with largely featureless chromatin. Thus, studies in this system have revealed the progression of genome activation triggered by pioneer factors that initiate DNA exposure at regulatory elements and the establishment of chromatin domains, including heterochromatin, the nucleolus, and nuclear bodies. The de novo emergence of nuclear structures in the early embryo reveals features of chromatin dynamics that are likely to be central to transcriptional regulation in all cells.The control of gene expression in eukaryotes relies on how transcription factors and RNA polymerases manipulate the structure of chromatin. These interactions are especially important in development as gene expression programs change. Chromatin generally limits the accessibility of DNA, and thus exposing sequences at regulatory elements is critical for gene expression. However, it is challenging to understand how transcription factors manipulate chromatin structure and the sequence of regulatory events. The  Drosophila in the 1980s reformulated our concept of development2. Later studies in other organisms revealed that the genetic control of development is conserved across eukaryotes, with common regulatory principles. A key issue is how transcription factors interact with the chromatin packaging of the genome for differential gene expression, even though the genome in different cell types is identical. Just as exceptional features of Drosophila embryogenesis provided a productive setting for identifying developmental regulators, studies in early embryos are now unraveling long-standing issues of fundamental mechanisms of the chromatin control of transcription.Development in multicellular organisms relies on differential gene expression to direct cell proliferation and differentiation. The identification of conserved transcription factors in genetic screens for embryonic defects in Drosophila embryogenesis are now being exploited to address this gap.The challenge in understanding transcriptional mechanisms is ordering molecular events and dependencies in living cells. Chromatin features have been correlated to gene expression and repression in many cell types, but it is more difficult to distinguish which features cause others and which actually regulate gene activity. Features of Drosophila females make a large egg that is loaded with proteins and untranslated mRNA. Fertilization by an incoming sperm triggers embryogenesis. A broad outline of chromatin changes in early embryogenesis is shown in 3. In its first hours, the embryo develops as a multinucleate syncytium with totipotent nuclei. Proliferative nuclear cycles occur synchronously and extremely fast, doubling nuclei every 10 minutes, so DNA replication, chromatin assembly, and mitosis occur at what must be close to maximal rates. These first rounds of nuclear replication and division use maternally supplied core histones and a variant histone H1 called bigH14 to duplicate chromatin. The rapidity of these divisions requires nearly simultaneous replication of the entire genome, with replication origins scattered at random roughly every 10 kb5. The speed of early divisions means that syncytial nuclei largely lack any of the localized chromatin features that characterize epigenomes in other cell types, but distinctions appear as nuclear cycles slow down, zygotic transcription begins, and the nuclei are partitioned to form the cellular blastoderm. In this way, the transition from the syncytial to the cellular embryo provides a tabula rasa for observing the onset and order of transcriptional programs and epigenomic specialization.Like most animals, 7. Widespread transcription of thousands of genes begins at nc14, referred to as the major ZGA, during the switch from maternal to zygotic control of morphogenesis. But how does zygotic transcription begin in the early embryo, and how does this alter the chromatin landscape?Zygotic transcription of a few genes begins around nc8, reaching a notable level of transcription from about 100 sex determination, cellularization, and early patterning genes around nc11, a time referred to as the minor zygotic genome activation (minor ZGA)8. At this time, developmentally regulated genes lack any nucleosome phasing around promoters or regulatory elements, but NDRs appear when transcription begins. At least three transcription factors have been implicated in driving zygotic transcriptional activation. The zinc-finger protein Zelda (Zld) is first translated beginning around nc8 from maternal mRNA and binds at thousands of sites11. These early sites include the promoters and enhancers of genes activated in the minor ZGA but also many regulatory elements of genes that will not be activated until the major ZGA. A second transcription factor implicated in early embryo transcription is the transcription factor GAGA factor (GAF), whose binding first appears in nc912. A second GAGA motif-binding protein called CLAMP is involved in the activation of some major ZGA genes13. These key transcription factors also cooperate with spatially localized transcription factors to establish the body plan of the embryo  where transcription factors bind. A small number of transcription-independent NDRs are detected in the nuclei of early syncytial embryos, probably due to intrinsic anti-nucleosomal sequence features19. For example, the mammalian pluripotency factors Sox2 and Oct4 bind DNA wrapped on the surface of a nucleosome and either shift its position or displace histones, thereby creating an NDR; exposure of this DNA then allows binding of secondary transcription factors. Thus, creating an NDR at binding sites is a key step for activating developmentally regulated enhancers and promoters, underlying the ability of these factors to reprogram cells from diverse types. These properties were first identified for the archetypal pioneer factor FoxA1, which initiates endoderm development19. Recent structural studies of pioneer factors docked onto nucleosomes in vitro have fleshed out factors that may bind specific DNA motifs even when wrapped around a histone octamer and how the binding of one factor may enhance the binding of a second20. However, the mechanism by which pioneer factors first bind sites in vivo has been more difficult to pin down. For example, a large fraction of binding sites for the FoxA1 factor depends on the cell type where it is expressed, implying that other bound transcription factors influence where FoxA1 can bind22. Close examination of FoxA1/2-binding sites in differentiating endoderm cells showed that these sites are partially accessible even before these factors are produced24. This makes it difficult to determine the relationship between site accessibility and pioneer factor activity, especially as the relevant cell stages in the early embryo cannot be easily observed.How do Zld and GAF bind chromatin in early nuclei? In many developmental systems, pioneer transcription factors are thought to convert inaccessible chromatin at binding sites into exposed DNA and thereby trigger new programs of gene expressionDrosophila embryo starts with a largely featureless epigenome, it provides insight into the chromatin sequence of pioneering. The early factors Zld, GAF, and CLAMP are pioneer factors in that each can bind nucleosomal DNA in vitro26. The creative use of mutations, engineered constructs, or microinjection into the syncytial embryo is deciphering the molecular activities of these pioneer factors  subunits have been tracked in this way, and nascent transcripts have been detected with fluorescent MS2 coat protein (MCP) fusions that bind engineered hairpins in mRNAs29. The embryonic syncytium can be manually injected without adversely affecting development, and studies have injected mRNA for fluorescent protein fusions with DNA-binding proteins to image specific DNA sequences or fluorescently-labeled antibody fragments to image specific histone modifications.In the 30. More sophisticated strategies are now applied in the embryo using inducible degradation components maternally loaded into the egg31, or optogenetic inactivation32 to trigger the elimination of factors during early embryogenesis. One particular method inspired by ‘anchors away’ technology in budding yeast uses the Drosophila Jabba protein to rapidly deplete GFP-labeled factors in the early embryo. Jabba normally localizes to lipid droplets in the early embryo, but a Jabba-anti-GFP nanobody fusion can sequester any target proteins outside of nuclei. Maternally loaded or injected mRNA encoding Jabba-anti-GFP nanobody has been used to acutely deplete histone methyltransferase proteins33, Zld, and Mxc34.Genetics and injection provide strategies to test function in the early embryo. Compounds that interfere with transcription, DNA replication, or chromatin remodeling can be injected. Since the early embryo relies entirely on maternally provided products until the beginning of the cellular blastoderm stage, many components can be altered by modifying the genotype of the mother. A range of genetic strategies are also available. For factors that are required only in the embryo, mutant mothers will produce eggs that lack a specific factor. In other cases, an interesting factor may also be important in other stages of development. Here, mitotic recombination schemes have been designed that produce homozygous mutant germline clones from heterozygous mothers, thereby giving eggs that lack the factor. Finally, for factors that are essential in the female germline, double-stranded RNA targeting a specific factor can be loaded into the egg, and this can accomplish knockdown in the embryo34. Zld accumulates in small foci in nuclei, and clusters of RNA polymerase II (RNAPII) and nascent gene transcripts appear adjacent to Zld foci35. These authors used an innovative sequestration method to rapidly deplete Zld from the embryo. This system uses an anti-GFP nanobody fused to the Jabba protein, which normally binds and sequesters target proteins in cytosolic lipid droplets in the embryo. Expression of this “JabbaTrap” fusion in embryos with a target GFP fusion protein thus sequesters the target away from chromatin33. Injection of JabbaTrap mRNA into embryos with a Zld-GFP fusion showed that Zld is required for the formation of RNAPII clusters. Intriguingly, blocking DNA replication with multiple different inhibitors greatly reduces the formation of Zld clusters and of RNAPII clusters. Clusters that do form are delayed and smaller. Thus, DNA replication appears to enhance the activity of Zld in early embryos.Live imaging has been used to follow the binding of Zld and transcription in the nuclei of early embryos. While earlier studies had shown that DNA replication initiates immediately after mitosis, and transcription slightly later, a recent preprint has detailed the link between replication and transcriptional initiation36. However, when Zld binds, nucleosomes around these sites acquire histone H3K27 acetylation8. CBP is the main H3K27 acetyltransferase, and eliminating the Drosophila homolog Nejire (Nej) in early embryos blocks RNAPII clustering and transcription34. This argues for a sequence of events as follows: first, DNA replication enhances the binding of Zld and clustering of RNAPII on chromatin; second, Zld stimulates histone acetylation by Nej; and, third, acetylation promotes RNAPII elongation34. But why would DNA replication enhance the activity of a nucleosome-binding pioneer factor? Perhaps the binding of Zld is enhanced by cell cycle-coupled protein modifications of the factor. Alternatively, early models for chromatin regulation in development proposed that the chromatin dynamics during DNA replication may provide a “window of opportunity” to bind transcription factors and start new transcriptional programs37. Nucleosome density and positioning are transiently reduced behind replication forks38. For some transcription factors, disorganized nucleosome positioning may delay binding, but others (like GAF) rapidly bind newly replicated DNA and position nucleosomes. Evidence that most Zld and RNAPII signals in early Drosophila embryos depend on DNA replication supports the idea of fast binding behind the replication fork. Importantly, a small fraction of Zld shows delayed binding in blocked embryos, and this might be due to the nucleosomal-binding activity of Zld. While this division between replication-dependent and nucleosomal-binding modes of Zld binding may be more extreme in the fast cycles of the early embryo, DNA replication may promote new transcriptional programming more generally.New nucleosomes behind DNA replication forks are assembled with acetylated H3 and H4 histones, and so while these modifications are enriched in early embryonic nuclei, most other histone modifications associated with transcriptional regulation are absent39. Indeed, both properties appear in the early Drosophila embryo. Live imaging of Zld protein shows that the factor releases from chromatin as mitosis initiates40, and the chromatin accessibility of Zld-bound sites is reduced as nuclei divide28. In contrast, GAF binds both interphase and mitotic chromatin, and mitotic GAF-bound sites remain accessible41. This difference appears to result from the longer residence time of GAF on chromatin that allows persistent binding to mitotic chromosomes. Thus, GAF fits the definition of a mitotic bookmarking transcription factor and appears to be functionally important, as imaging nascent transcription of a target promoter has shown GAF-dependent mitotically heritable expression41.The fast nuclear division cycle of early embryos has also revealed factor-specific distinctions for mitotic inheritance. While many transcription factors dissociate as mitotic chromatin condenses, a long-standing question in mammalian systems has been whether some factors persist in maintaining stable patterns of gene expression in cell lineages44. These can be understood in the framework that pioneering activity is a matter of degree, as transcription factors vary in affinity from site to site and cooperatively interact with other chromatin proteins45. However, one distinct class comprises about 2,000 promoters for housekeeping genes that are activated in the major ZGA but lack binding by any known pioneer factors. Instead, these promoters are enriched for the H2AV histone variant (the Drosophila ortholog of H2A.Z and H2A.X variants)46. H2AV is deposited at these promoters well before these genes are zygotically expressed by a conserved SWR1-type chromatin remodeler called Domino. While the elimination of Domino from the early embryo prevents H2AV accumulation and gene activation, chromatin accessibility is not altered. It remains to be determined whether these sites are targeted by pioneering core promoter factors or whether intrinsic nucleosome-positioning features of their DNA sequences make them accessible.There are additional transcription factors that activate genes in the major ZGAde novo establishment of nuclear organization in the early embryo includes the appearance of nuclear bodies and distinctive chromatin regions, including the histone locus body (HLB), the nucleolus, and heterochromatin. Observation of how these features appear reveals the steps in establishing phase-separated compartments within the nucleus. The HLB first appears in nc11 when the histone genes are transcribed from a gene array on chromosome 2, but DNA-binding factors — including the CLAMP pioneer factor and the NPAT homolog Mxc — first arrive at the histone genes one cycle earlier in nc1047. This nuclear body appears to be anchored by a DNA-bound seed, while processing factors may localize into the HLB through interactions with histone mRNA48. A similar sequence drives the formation of the nucleolus, where transcription of the rDNA genes from arrays on the X and Y chromosomes begins in nc11, and processing factors appear in nc1349 . In Drosophila, regulatory elements called Polycomb Response Elements (PREs) are required for gene silencing, and PREs first become accessible in nc1152. Many PREs are binding sites for GAF, so the general mechanism of pioneering activity initiating zygotic gene expression also applies to the establishment of developmental gene silencing.Observations in the early embryo have also detailed the temporal relationship between histone modifications and gene regulation. Chromatin in the early embryo is globally hyperacetylated because of replication-coupled nucleosome assembly, but active modifications, such as H3K4 and H3K36 methylations, first appear after gene transcription is initiated in the major ZGA33. More widespread heterochromatic features, including delayed replication and then HP1 binding, are apparent in nc13 and nc1454. Intriguingly, the embryonic establishment of heterochromatin on a specific satellite block depends on its maternal presence, suggesting that maternally-loaded RNAs from this satellite are required to establish heterochromatin in the embryo55. Similarly, maternal PIWI-interacting RNAs (piRNAs) may work on other repetitive sequences in the nucleus56. These dependencies may be distinct from the activities that maintain mature heterochromatin in later cell types.The establishment of heterochromatin in the embryo may operate by a different mechanism. In most cell types, repetitive transposons and short satellite repeats replicate very late in the S phase and become wrapped in nucleosomes marked by histone H3K9 methylation, comprising a repressive heterochromatic state. In the fast cycles of the early embryo, all sequences replicate at the same time, but repetitive sequence blocks begin to show heterochromatic properties at different times in later stages. Some satellite blocks become compacted in nc8 and gain H3K9 methylation in nc12, which then accumulates to high levels in successive cycles as interphases get longer57. This organization is thought to result from both cohesin binding that constrains the chromatin fiber and from non-specific associations between decondensed transcribed chromatin and inactive chromatin. As the early Drosophila embryo is transcriptionally silent, this stage provides a natural setting to track the acquisition of nuclear organization. Mapping of 3D organization in Drosophila embryos revealed that only a few boundaries are present in very early embryos, and these coincide with constitutive H2AZ-containing promoters46. Additional TADs and compartments appear in nc8 when Zld binds to chromatin59. This order of events demonstrates that factor binding alone can create domains and that transcription is not needed. Factor binding at TAD boundaries and at looping elements creates a framework of invariant cell type-independent domains in embryos, but only a subset of these interactions are productive in specific cell types61. Direct imaging of individual nuclei in the Drosophila embryo has further refined our understanding of nuclear organization. While contact maps have led to the impression of extensive 3D organization within nuclei, tracing chromatin paths64 or imaging the juxtaposition of transcribing genes35 shows that 3D organization in individual nuclei is very diverse, with structures detected by contact mapping being rare and transient. This places important limits on the functional significance of higher-order structures for gene expression, where 3D interactions appear to be a pre-condition for gene activation but do not determine it.The power of observation in the early embryo is now being applied to deciphering how the 3D organization of the nucleus is established. Chromatin in the nucleus displays a hierarchy of interactions, where loops between regulatory elements organize genes within topologically associated domains (TADs), which are further aggregated into inactive and active compartmentsDrosophila embryo has revealed that the binding of a small number of key DNA-binding proteins can trigger the organization of much of the nucleus, which successive gene activity elaborates. The sequence by which nuclear features emerge implies that they are consequences of “sticky” weak electrostatic interactions. These issues even apply to universally accepted concepts, such as how enhancers act on promoters to activate transcription. While textbooks describe that promoters and enhancers loop together in 3D space to form an activating structure, such contacts are rare and transient in direct imaging. How can transient contacts promote the slower process of transcriptional activation? One class of models suggests that an enhancer leaves a long-lasting mark on promoters, perhaps as histone modifications or stabilizing bound factors, and the persistence of these effects at promoters stimulates transcription62. More radical models propose that enhancers produce diffusible molecules that move across nuclear distance to activate promoters65. The ability to resolve the sequence of nuclear events in time and space in the early embryo holds promise in understanding these fundamental issues in gene regulation.Whereas the core questions in chromatin biology were posed many years ago, the intricacy of many eukaryotic systems has complexified the answers. The blank slate of chromatin in the early embryo has provided a simple setting for probing the mechanics of how transcription factors first interact with chromatin, how phase-separated bodies within the nucleus are first established, and how 3D nuclear organization comes to be. The