Unlike most transcription factors (TF), pioneer TFs have a specialized role in binding closed regions of chromatin and initiating the subsequent opening of these regions. Thus, pioneer TFs are key factors in gene regulation with critical roles in developmental transitions, including organ biogenesis, tissue development, and cellular differentiation. These developmental events involve some major reprogramming of gene expression patterns, specifically the opening and closing of distinct chromatin regions. Here, we discuss how pioneer TFs are identified using biochemical and genome-wide techniques. What is known about pioneer TFs from animals and plants is reviewed, with a focus on the strategies used by pioneer factors in different organisms. Finally, the different molecular mechanisms pioneer factors used are discussed, highlighting the roles that tertiary and quaternary structures play in nucleosome-compatible DNA-binding. Transcription factors (TFs) are DNA-binding proteins that read genomic information to control gene expression in all organisms ,2,3,4,5.The pioneer factor concept originated from in vivo footprinting studies, where researchers sought to determine which TFs were the first ones to bind a tissue-specific enhancer during embryonic development ,11. Two Pioneer factors were originally identified as master regulators of cell fate and their ability to reprogram cell fate has been investigated at the molecular level. The extensive reprogramming of gene regulatory networks triggered by pioneer factors requires the opening and/or closing of different chromatin regions and the binding of nucleosomal DNA . Indeed,For in vivo evidence of pioneer activity, genome-wide binding experiments, and correlation with chromatin states at the TFBS have been performed for different pioneer factors . Due to ChIP-seq has long been considered the technique of choice to map TFBSs in the genome for a given TF. Briefly, the protein of interest is cross-linked with the DNA, and the TF-DNA complex is purified through immunoprecipitation (IP) with an antibody. Subsequently, the protein-associated DNA is subjected to sequencing to identify the genomic regions bound by the TF. When compared with chromatin accessibility assay profiles (see below), it is possible to derive information with respect to whether or not a TF can target closed or poorly accessible chromatin, a key feature for pioneer factors. ChIP-seq has been successfully used for many TFs, and several ChIP-seq variant methods have been developed, which are capable of dealing with low-input materials and giving a high signal-to-noise ratio, among other advantages. Such variants includes ORGANIC  , ChEC-seThe building blocks of chromatin are the nucleosome core particles, each consisting of approximately 147 base pairs of DNA wrapped around a histone octamer composed of four different core histones, generally, H3, H4, H2A, and H2B. Nucleosomes are arranged into ‘beads on a string’-like structures and are further compacted into highly condensed structures that confer transcriptional silencing ,68,69,70Nuclease-based assays, such as DNase-seq ,72,73 anAs alternative methods of determining chromatin accessibility, FAIRE-seq  and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) have been recently developed. FAIRE-seq has been used frequently due to its simplicity and lack of enzymatic digestion . In brieIn higher eukaryotes, the genome is organized into hierarchical folding of chromosomes in highly ordered three-dimensional structures. As revealed by chromosome conformation capture (3C) assays, chromosomes are divided into territories of A (active) and B (inactive) compartments ,83,84,85In the last few years, many chromatin-conformation capture methods have been developed, such as 3C, 4C, 5C, Hi-C  and otheTransgenic experiments provide another means of assessing pioneer activity of a given TF. In loss-of-function experiments, for example, removal of a pioneer factor should directly reduce the accessibility of its target regions, whereas in gain-of-function experiments, ectopic expression of the factor should increase the accessibility of these regions. In many pioneer factor studies, characterization of loss-of-function and gain-of-function lines is usually applied. For example, a recent study by Jacobs et al. suggested that GRAINY, a highly-conserved TF with essential roles in epithelial cell-fate specification and wound healing in animals, is a pioneer factor. The authors showed that deletion and ectopic expression of GRAINY causes loss and gain of DNA accessibility, respectively, suggesting pioneer activity . Alternaebf1 mutant cells, they manage to elucidate the hierarchy of events. They showed that DNA demethylation is in fact preceded by the formation of chromatin accessibility, which is again preceded by EBF1 occupancy. In another study, Mayran et al. used an inducible version of PAX7 and studied the dynamics of the chromatin in a similar, time-resolved manner [Inducible systems, combined with time point experiments, are a robust way to investigate pioneer activity of a given factor, but are not high-throughput and require expertise to be properly performed. Among the most widely used inducible systems are estrogen receptor or glucocorticoid receptor-mediated systems, which have been applied for pioneer activity determination of, e.g., EBF1 , PU.1 4, PAX7 3, Activatd manner . StrikinAs described above, genome-wide techniques are a powerful tool for the investigation of pioneer factors. However, it is important to carefully interpret the data. Assessing the ability of a certain factor to bind nucleosomal DNA can be achieved by combining ChIP-seq or its variants with chromatin accessibility assays. However, overlapping peaks of protein-DNA binding and closed chromatin do not indisputably prove that a factor can bind closed chromatin, as these interactions may be indirect. In addition, samples must be taken at the same developmental stage from the same tissue/cell population to avoid misinterpretation of TF binding and chromatin status. Not being able to detect a perfect overlap between chromatin accessibility and DNA binding does not necessarily indicate that a TF is not a pioneer factor, either. A recent study on GR-responsive enhancers showed that nucleosomal regions can sometimes show hypersensitivity to DNase I, in contrast to the idea that hypersensitivity reflects absence of nucleosomes . A studyThe idea of ‘pioneer factors’ was originally proposed based on functional studies that showed that the TFs FOXA1 and GATA4 were able to potentiate liver organogenesis from endoderm cells. This functional feature, namely, the ability to program or reprogram cell fate, was associated with the activation of previously silent target genes. In the last decade, many key TFs involved in cell fate specification have been identified as pioneer factors in mammals. These pioneer factors act as master regulators of major cellular events, including cell fate programming from embryonic cells to differentiated cell types, re-programming from somatic cells to pluripotent cells, and direct cell conversion or trans-differentiation, such as from fibroblasts to muscle cells .The differentiation of embryonic cells to distinct cell types in early embryonic development requires a dramatic reprogramming of gene expression patterns. Pioneer factors play a critical role in establishing competence for many different cell fate specification programs, for example, PAX7 in pituitary melanotrope development and PU.1 in myeloid and lymphoid development, among many others . Cell tyArf, a tumor suppressor [Pioneer TFs are also able to directly reprogram cells by switching somatic cells to a different type of somatic cell without passing through an intermediate pluripotent stage. For example, the combination of PU.1 and C/EBPα/β is sufficient to convert fibroblasts to macrophage-like cells , in whicppressor . In thesFLOWERING LOCUS C (FLC) [FLC is a floral repressor which, during cold winters, is epigenetically repressed by Polycomb repressive complex 2 (PRC2) in a process called vernalization [Our knowledge of pioneer activity in plant species is much more limited. A few TFs have been described as potential pioneer factors in plants, including LEC1, LEAFY (LFY), APETALA1 (AP1), and SEPALLATA3 (SEP3) . LEC1 is C (FLC) . FLC is lization ,109. Thelization ,110,111.lization ,110,111.N-terminal domain, and a C-terminal novel helix-turn-helix DNA binding domain (DBD). The SAM domain does not affect DNA binding in vitro, but is required to fully complement the lfy-12 mutant, suggesting that oligomerization is required for LFY function and that it potentially plays a role in pioneer function. ChIP-seq experiments performed on 2-week-old seedlings expressing either a LFY or LFY SAM-domain mutant impaired in oligomerization revealed a strong reduction of binding when the SAM domain was impaired. Comparison of ChIP-seq data with DNase-seq data performed in the same conditions suggested that LFY was able to bind to closed chromatin regions, and that the SAM domain was required for this binding [LFY acts as a master regulator of flower development  and was  binding . In addi binding , which aThe MADS-box family TFs, AP1 and SEP3, have also been described as potential pioneer factors . These TPioneer TFs from animals and plants share common characteristics, namely, the ability to bind closed regions of chromatin and to trigger the opening of these regions, rendering them competent for the binding of other factors and/or gene expression. Different mechanisms have been proposed for pioneer factor-DNA binding in nucleosome-rich regions of chromatin. These include the ability to mimic linker or core histones, the capacity to bind in a nucleosome-compatible manner to a single face of the DNA, and the use of oligomerization to increase binding affinity to outcompete nucleosomes at their cognate binding sites. Upon successful binding, opening closed regions of chromatin may occur either through the direct displacement of nucleosomes or through the recruitment of chromatin-remodeling proteins and complexes.A number of identified pioneer TFs possess histone-like folds, suggesting that histone mimicry may play an important role in pioneer function . For exaAnother proposed mechanism distinct from histone-fold mimicry is single-side DNA binding that is seen in the mammalian pioneer structurally unrelated TFs, OCT4, SOX2, and KLF4 . In planOligomerization and cooperative binding, which increases DNA-binding affinity, has been hypothesized to play an important role in allowing pioneer factors to bind their cognate sites even in relatively inaccessible nucleosome-rich chromatin regions. For example, the mammalian pioneer factors OCT4, SOX2, and KLF4 are all able to oligomerize, increasing their binding affinity. A similar mechanism is likely used in plants. As mentioned previously, Arabidopsis LFY requires oligomerization activity in order to bind to low affinity LFY binding sites in closed chromatin regions . SimilarWhile histone mimicry can result in the direct displacement of histones, some pioneer factors open closed chromatin regions via the recruitment of chromatin remodelers. In mammals, one of the common chromatin remodelers that can be recruited by several pioneer factors, in most cases through direct physical interactions, is the ATPase BRG1 of the BAF complexes. For example, GATA1 has been shown to recruit BRG1 to closed chromatin regions and create accessible sites for secondary factors, such as TAL1, for downstream transcriptional activation, required for differentiation of human hematopoietic stem cells to erythrocytes . FurtherBesides binding to nucleosomal DNA, pioneer factors have been shown to be able to bind sites that are transcriptionally silenced by DNA methylation. DNA-methylated binding sites, and in particular, CpG methylated sites, are often regarded to be inaccessible to canonical transcription factors . HoweverAfter the binding and opening of closed chromatin regions, some pioneer factors are able to facilitate the creation of long-term accessible chromatin regions to non-pioneer factors. To achieve this, pioneer factors promote the deposition of permissive histone modifications, such as H3K27ac3, and/or elimination of repressive histone markers, such as H3K27me3. For example, pioneer factor TCF-1 can erase H3K27me3 and H3K9me3 repressive marks near TCF-1 bound sites during T cell development in mice  Table 1Table 1. Up to now, no pioneer factor has been discovered to be able to have unrestricted access to all states of heterochromatin. In fact, pioneer factors exhibit cell specificity, being able to bind sites in certain cell types, but not in others . For thePioneer factors are highly important in the development of eukaryotes. What strategy they use to overcome the difficulty of activating silenced genes in heterochromatin regions is a fundamental biological question. In the last few years, the knowledge of the mechanisms of action of these factors in human and animal systems has grown tremendously, facilitated by the rise of genome-wide techniques. It has become clear that different pioneer factors have different modes of action—how they interact with closed chromatin , the way they allow opening of the chromatin (histone-mimicry or recruitment of chromatin remodelers), and how they help establish stable epigenetic marks.While the majority of research on pioneer factors has been performed on mammalian systems, pioneer factors are starting to be identified in other eukaryotes. The idea of pioneer factors in plants only emerged several years ago. Interestingly, amongst the currently proposed plant pioneer factors, AP1, SEP3, LFY, and LEC1, only the latter shows homology to an animal pioneer factor. AP1 and SEP3 are MADS-box proteins, a protein family that does not appear to act as pioneer factors in animals, and LFY is an orphan protein that can only be found in plants. On the other hand, it appears that most of the animal pioneer factors belong to TF families that are absent from plants. For example, the forkhead box proteins that the different FOX pioneer factors belong to, and the Paired-Box TFs that include PAX7, are not found in plants.Arabidopsis [Arabidopsis [Arabidopsis genome [Some mammalian pioneer factors belong to large families of TFs that are also present in plants, but they have not yet been identified as having a pioneer role. These factors include MYOD1 and ASCL1 of the bHLH TF family (more than 160 members in bidopsis ) and ZELbidopsis ). The GAs genome , and ares genome ,143. Meas genome ,134. To Many animal pioneer factors have a function in embryogenesis. When it comes to putative plant pioneer factors, it is important to realize that plants and animals differ greatly in this process. All organs are present after embryogenesis in animals, but organogenesis takes place throughout the plant life cycle. The putative pioneer factors in plants currently described are all involved in the developmental transition from vegetative to reproductive development. This switch requires fine-tuned remodeling of the chromatin architecture, as it is essential for the survival of the plant to not flower prematurely. Therefore, it is not unlikely that plants have evolved different pioneer factors to engage in important phase transitions during their lifetime. Hence, plant and animal pioneer factors might exhibit different modes of action in terms of both structure and function, and whether this is indeed the case will require further research.


Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths.Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in   •Chromatin modifiers Kismet and Trr limit intestinal stem cell proliferation•Kismet and Trr colocalize at transcriptionally active regions and co-regulate genes•Cbl is a target gene of Kismet and TrrEGFR negative regulator •Kismet and Trr limit EGFR signaling in ISCs, preventing tumor-like ISC accumulation Drosophila intestinal stem cell (ISC) whole-genome profiling of chromatin modifier Kismet/CHD7/CHD8 binding to DamID reporters of different chromatin states and demonstrate that Kismet and Trr/MLL3/4 are regulators of ISC self-renewal by limiting EGFR signaling.Stem cell proliferation control is essential to maintaining tissue homeostasis. Gervais et al. compare  Regulation of stem cell proliferation rates is critical in adult tissues, which need to maintain basal renewal and undergo damage-induced regenerative responses. Consequently, the dysregulation of stem cell proliferation can have pathological effects. Ample evidence now supports a functional link between the deregulated proliferation of stem cells and cancer initiation, as well as metastatic progression . InteresDrosophila midgut, which is maintained by around 1,000 multipotent intestinal stem cells (ISCs). Most ISC divisions lead to asymmetric daughter cell fates, resulting in a self-renewed ISC and a sister enteroblast (EB) cell (A useful model to investigate adult stem cell regulation is the EB) cell A. A majoEB) cell . In respEB) cell . Of primEB) cell . MoreoveEB) cell . EvidencEB) cell , though kismet, similar to the chromodomain-containing chromatin remodeling factors CHD7 and CHD8. Mammalian CHD7 is associated with transcriptionally active genes at enhancers, super-enhancers, and promoters where it can both activate and repress transcription , Kismet was proposed to promote transcription elongation  . Moreove cancers . CHD8 muisorders . In Drosd memory . HoweverCbl, a negative regulator of EGFR signaling. Our data therefore demonstrate that the chromatin regulators Kismet/CHD7/CHD8 and Trr complex/MLL3/4 function together to limit basal levels of ISC proliferation and identify Cbl as a key downstream target gene allowing control of EGFR signaling.Here, we demonstrate that Kismet is an important regulator of ISCs, essential to limit basal levels of ISC proliferation. DNA adenine methyltransferase identification sequencing (DamID-seq) of Kismet compared with DamID reporters of different chromatin states, including activated (Brahma and RNA Pol II) and repressed states , demonstrated that Kismet preferentially localized to transcriptionally active regions of the genome and to developmental enhancers. In addition, our data suggest that Kismet acts in ISCs with the H3K4 monomethyltransferase, Trithorax-related complex . We find that Trr and Kismet co-localize in the genome and co-regulate the transcription of many genes, including We screened for EMS-induced mutations affecting ISC activity and intestinal homeostasis  and found one line (10D26) that showed an increase in the size of mutant clones generated in the midgut of adult flies B and 1C kismet gene  indicated that 10D26 harbored a lethal mutation in kismet. kismet encodes a conserved chromatin remodeling factor, similar to CHD7 and CHD8 in mammals. kismet encodes a long (Kismet-L) and short (Kismet-S) isoform, with only Kismet-L containing two chromodomains and an SNF2-like ATPase/helicase domain, required for nucleosome remodeling activity  encompassing the genomic locus containing kismet fused to a FLAP tag-encoding sequence  also showed a partial rescue of the mutant phenotype  and EE cells (Pros), though they made more per clone, consistent with larger clone sizes , e midgut A and 2B,kismet mutant clones had at least one mitotic cell, and clones with more than one dividing cell were detected  as a marker for mitotic cells. 7.6% of the control clones had at least one and never more than one mitotic cell per clone C–2E′. Indetected  and founfraction G–2I. Togkismet activity is required, we first assessed its expression profile in the intestine. An antibody recognizing the C-terminal part shared by the short and long forms of Kismet showed enrichment in cells positive for Escargot (esg), a marker of ISCs and EBs, and in EE cells compared to ECs  by shifting to 29°C to inactivate a temperature-sensitive GAL80 (GAL80ts). At 18°C, control guts showed isolated Delta+ ISCs, whereas those with induced kismet mutant clones showed clusters of Delta+ ISCs  or expression of capicua (cic), a downstream repressor of EGFR target genes , Histone H1, or HP1 (a reader of histone H3K9me3) . Global in flies H and 4B.in flies . We thenin flies C or within flies . A majorin flies  and 14.5in flies E. Consisin flies A–S5A″. TOur data suggested, however, that Kismet more strongly overlapped with activated chromatin states, visualized on UMAP by Kismet enrichment with RNA Pol II and also to some extent with Brahma, a SWI/SNF chromatin remodeling factor associated with histone H3K27ac C. 85.1% esg, sox21a, Delta, and spdo; We then assessed how Kismet localized relative to active genes and enhancers. We established a list of expressed or “active” ISC genes based on Dam-RNA Pol II gene occupancy, a good proxy for gene expression  STAR Me. 4,539 gInterestingly, while Kismet showed a strong enrichment for active genes J, it wasWe then investigated the binding profiles of Kismet and RNA Pol II at previously published enhancers defined in S2 cells as being either “developmental” or “housekeeping” . Intereskismet inactivation in the gut had a similar effect on limiting Histone H3K27me3 but found no detectable global increase in H3K27me3 in kismet mutants  to sort adult ISCs  of controls (white RNAi) or those expressing RNAi against kismet or trr and performed RNA-seq  in kismet RNAi-expressing clones rescued clone size and accumulation of Delta+ cells supporting that Cbl acts downstream of Kismet to regulate ISC self-renewal  and UTX   . These d and lpt L–S7N′, bt clones M–S6N′ orre found H–S7K′. Ire found . Furtherre found . Thus, wCHD7 and MLL4 mutations on human development, both CHD7 and MLL3/4 complex components are mutated in cancers. CHD7 is found as a highly expressed fusion protein in small cell lung cancers .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Allison Bardin , kis1 (#431), UAS-kis-RNAi (#36597) for UAS-kismet RNAi #34908 for kismet knockdown RNA-seq condition), UAS-lpt-RNAi (#25994), UAS-trr-RNAi (#29563), UAS-utx-RNAi (#34076), FR82B trxE2 (#24160), FRT19A trrB encoding a putative truncated 512-aa protein (#57138), UAS-EGFRDN (#5364), UAS-bskDN (#6409), UAS-yki-RNAi (#34067), UAS-InRDN (#8253), UAS-domeRNAi (#34618), UAS-ash1-RNAi (#31050 and #36130), UAS-brm-RNAi (#31712), UAS-Cbl-RNAi (#27500), UAS-GFP-E2F1-230, UAS-mRFP-CycB1-266 (#55118), 10XSTAT92E-GFP (#26198), CycE-lacZ (#30722), , UAS-set1-RNAi (#40682), UAS-cic-RNAi (#103805) , kisLM27 and kisEC1   GBE-GAL80 tubGAL80ts     , esg-GAL (esgts) , esg-GALREGAL80) , MyoIAGAMyo1Ats) . prosvoi(prosts) .kislocus construct containing 524 bp upstream to 12 kb downstream of the kis gene was generated starting from Drosophila BAC (P[acman] BAC CH321-35E09) that was further altered by recombineering to reduce the genomic size and to add a FLAP cassette N-terminally . The FLAP cassette is composed of green fluorescent protein (GFP), S- and Flag-affinity tags separated by PreScission- and TEV- protease sites. Transgenic flies were generated at Bestgene, Inc. by injection of attP-9AVK00013. To generate UAS-kisL and UAS-kisS transgenic flies, kis-RA and kis-RB cDNA were respectively amplified from a midgut library and then inserted into the pUASPattB plasmid and tagged with both a 6xHIS N-terminal and a FLAG C-terminal cassette for kis-RA and only by a FLAG C-terminal cassette for kis-RB. Transgenic flies were generated at Bestgene, Inc. by injection of attP-3BVK00033 embryos. The mutation of Chd7 K999R, a residue in the highly conserved ATP binding motif of SNF2 superfamily proteins, was shown to prevent its ATPase catalytic activity  on 3-day-old adult females and were dissected 5, 9, 10, 12 or 30 days after heat shock.Clones were generated with the Mosaic Analysis with Repressible Cell Marker (MARCM) technique . The folts clones were generated using the following stock: w P[hs-FLP] P[pTub-GAL4] P[UAS-nlsGFP]; FRT40A P[pTub-GAL80]; P[pTub-GAL80ts]. Crosses were maintained at 18°C, before and after 35 minutes heat shock clone induction at 36.5°C in 3-day-old adult females. 10 days AHS, temperature was shifted to 29°C for 3 days before dissection to allow transgenes expression (UAS-GFP and UAS-NAct).MARCMkismet RNAi we used the temperature sensitive inducible UAS-GAL4/GAL80ts system. Crosses and adults were kept at 18°C, the GAL80 permissive temperature. 3-day-old flies were shifted to 29°C for 2, 3 or 10 days to induce RNAi expression.For temporal cell type-specific expression of ECD staining, guts were fixed for 15 min in 4% formaldehyde/heptane followed methanol treatment and rehydration in PBT as described in ); anti-GFP , anti-DsRed , anti-Sanpodo , anti-Notch ECD C458.2H , anti-ßGAL , anti-Prospero , anti-Pdm1 , anti-PH3 , anti-Kismet DK20 , anti-Utx, anti-Trr, anti-Lpt and anti-H3K4me1 , anti-dIn DamID, the fusion of Dam methyl transferase to a DNA associated protein allows the methylation of surrounding GATC sites of DNA, which can be specifically sequenced . In targDrosophila melanogaster reference genome version 6 using bowtie2 . Genes were classified as bound by a protein if 2 consecutive GATC sites within the gene body were occupied with an adjusted p-value < 0.01.Peaks were called by merging 2 or more consecutive significant GATC sites  across annotated transcripts using “polii.gene.call” script and false discovery rates (FDR) were assigned . Genes wAll analysis has been done on Galaxy .2 values estimated by DESeq2. To evaluate the embedding we plotted the log2 value for each GATC and each chromatin protein in the UMAP coordinates. We explored effect of varying the n_neighbors, min_dist, n_components and metric parameter and note that varying the parameters results in very similar maps. The parameters used are n_neighbors=30, min_dist=0.0, n_components=2, random_state=42, metric='canberra'.A 2D UMAP embedding  was creaUAS-GFP with UAS-white-RNAi (control), or with UAS-kis RNAi or with UAS-trr-RNAi under the control of esgts NREGal80 were shifted from 18°C to 29°C for 2 days to induce RNAi expression. For each biological replicate  midguts from 100 females were dissected in PBS before FACS sorting isolation of ISC GFP+ cells followed by RNA isolation and amplification as described in  using Salmon  were scored except for https://github.com/bardin-lab/kismet-analysis. DamID-Seq and RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO). The accession number for DamID-Seq and RNA-Seq data reported in this paper isGSE128941.Galaxy workflows, IPython notebooks and UMAP code used in the analysis are available at


A complex interplay between chromatin and topological machineries is critical for genome architecture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism.scc1-73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the − 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1-73 cells, downregulated genes are enriched in promoters with short or no nucleosome-free region (NFR) and a fragile “nucleosome − 1/RSC complex” particle. These results, together with a preferential increase in the occupancy of nucleosome − 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1-73 upregulated genes are enriched in promoters with an “open” chromatin structure and are mostly at cohesin-enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome.We have used genome-wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin-mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins.The online version of this article (10.1186/s13072-019-0293-6) contains supplementary material, which is available to authorized users. The first level of structural organization of chromosomes is the chromatin fiber, in which DNA is assembled into regularly spaced nucleosomes through the coordinated activity of histone chaperones, chromatin assembly factors and nucleosome modifiers and remodellers. Assembly of replicated DNA is coupled to the replication fork and occurs by the deposition of both newly synthesized and parental histones, which are distributed randomly between the sister chromatids . In addiChromatin fiber permits several levels of compaction of DNA into the nucleus, depending on the nuclear functional state or the cell cycle phase. Although the exact molecular structure behind compaction is still unclear, current models suggest that the packaging of chromatin requires dynamic and regulated interactions between irregularly folded fibers of nucleosomes, leading to a meshwork of intramolecular contacts . The comIn budding yeast, cohesins are loaded at chromatin by the cohesin loader complex Scc2/Scc4 during G1 and early S phase in a non-stable conformation that promotes topological and non-topological interactions  through dynamic turnover –10. AfteNucleosomes seem to have an inhibitory effect on cohesin binding. The Snf2-related complexes RSC and Irc5 in yeast, and SNF2h in humans are required for cohesin binding to chromosomes , 29, 30.To address the connection between chromatin structure and cohesin function, we have now studied whether disrupting one affects the other. For this, we allow cells to progress from G1 to mitosis under conditions of histone depletion and/or lack of cohesin activity. Defective chromatin positioning by histone depletion had little or only region-specific effects on the accumulation and distribution of the major cohesin peaks. In contrast, the lack of cohesin activity affected the primary structure of chromatin at specific genomic regions, preferentially promoters. Critically, genome-wide analyses revealed a major role for cohesins in transcription regulation that is associated with the promoter chromatin architecture and location of the regulated genes.SCC1 (scc1-73). The α-kleisin subunit Scc1 forms the tripartite ring-like structure with the SMC  subunits Smc1 and Smc3 in the cohesin complex. The scc1-73 allele encodes a mutant protein (S525 N) that loses its ability to interact with Smc1/Smc3 at 37 °C and thereby causes cohesin inactivation , considering a maximum gap of 250 bp and a minimum run/length of 30 bp.High-resolution (5-bp) ChIP-on-chip analyses were performed with two biological samples for wild type and scc1-73 were constructed and sequenced at the Genomics Core Facility of CABIMER. For this, strand-specific total RNA-seq libraries from 100 ng RNA samples were prepared using the TruSeq® Stranded mRNA Library prep kit (Illumina). Indexed libraries were pooled and sequenced on Illumina NextSeq 500 using paired-end chemistry with 75 bp read length to a depth of approximately about 37 million reads per library. Raw reads were filtered and trimmed with FASTQ toolkit 1.0.0 and assessed using FastQC 1.0.0 by BaseSpace Sequence Hub Illumina website. FASTQ data were uploaded to the Galaxy web platform for further analyses [S. cerevisiae genome version sacCer3 using the HISAT2 aligner [RNA was extracted using a standard hot acid phenol extraction protocol  and purianalyses . Briefly aligner  and filt aligner . Differe aligner  and the  aligner .http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the accession numbers GSE121067 (MNaseI-seq), GSE121004 (ChIP-on-chip) and GSE125258 (RNA-seq).Nucleosome profiles and cohesin peaks along the genome were visualized using the Integrative Genomics Viewer (IGV) . All datAnalyses of the genome distribution and overlapping of dynamic nucleosomes, cohesin peaks and gene elements were performed with the tools of the Galaxy web platform  . Other sAdditional file 1: Table S1. Comparison of the number and percentage of nucleosomes altered after histone depletion (in t::HHF2 cells) or cohesin inactivation (in scc1-73 cells) in the indicated genomic regions relative to the whole genome. A hypergeometric test was used to determine the probability to obtain the indicated percentages of altered nucleosomes at the indicated genomic regions if their distribution along the genome were random. Significantly higher (green) and lower (red) percentages are highlighted. *rDNA is analyzed as a single copy.Additional file 2: Table S2. Comparison of the number and percentage of nucleosomes altered after histone depletion (in t::HHF2 cells) or cohesin inactivation (in scc1-73 cells) in the indicated genomic regions relative to the whole genome. A hypergeometric test was used to determine the probability of obtaining the indicated percentages of altered nucleosomes at the indicated genomic regions if their distribution along the genome were random. Significantly higher (green) and lower (red) percentages are highlighted. *rDNA, analyzed as a single copy.Additional file 3: Table S3. Misregulated genes in ssc1-73. Lists of up- and downregulated genes are shown.Additional file 4: Table S4 List of misregulated genes from additional studies used for comparative analyses in Table 2.Additional file 5: Table S5. Cohesin distribution in wild-type and histone-depleted cells at the indicated genomic regions. A peak of Scc1 is defined as a DNA fragment with continuous Scc1 signals that are both positive (relative to the untagged strain) and with a p < 0.05. A peak signal was calculated as the sum of these positive signals. Genomic regions with Scc1 and peaks of Scc1 at a particular genomic region are defined by at least 1 bp overlapping.Additional file 6: Table S6. The effect of cohesin inactivation on chromatin structure is more severe in cohesin-associated regions. The probability that scc1-73 alters nucleosomes in Scc1-binding IGR or Scc1-binding ORF if the altered genomic regions in scc1-73 cells were randomly distributed was determined by a hypergeometric test. The rate between the observed and expected frequencies of common genomic regions is also shown. The number of regions in each case is indicated in parenthesis.Additional file 7: Table S7. Saccharomyces cerevisiae strains (a) and oligos (b) used in this study.Additional file 8: Fig. S1. Effect of cohesin inactivation in scc1-73 cells on the chromatin structure of three different loci of histone-depleted cells. Nucleosome positioning analyses are shown for the indicated loci after MNase I digestion and indirect-end labeling of the indicated strains synchronized in G1 and released into fresh medium for 1 h at 37 °C until G2/M.Additional file 9: Fig. S2. Effect of histone depletion on cohesin binding and distribution. a Cohesin distribution at different regions of chromosomes II and V in wild-type and t::HHF2 cells that have been synchronized in G1 and released into fresh medium until G2/M, as determined by ChIP-on-chip analysis against HA-Scc1. b Probability that IGR that had either lost or gained cohesins after histone depletion would overlap with IGR with altered nucleosomes in t::HHF2 cells or in both t::HHF2 and scc1-73 cells if they were randomly distributed, as determined by a hypergeometric test. The rate between the observed and expected frequencies of common IGR is also shown. The number of IGR in each case is indicated in parenthesis. nd, not determined.Additional file 10: Fig. S3. Preparation and analysis of the nucleosomal DNA used for MNaseI-seq. a Generation of mononucleosomes in the indicated strains after partial digestions with MNase I. The DNA purified for DNA-seq is marked in red. b, c Electrophoretic (b) and electropherogram (c) analyses of the purified nucleosomal DNA.


Cohesin is a conserved, ring-shaped protein complex that topologically embraces DNA. Its central role in genome organization includes functions in sister chromatid cohesion, DNA repair, and transcriptional regulation. Cohesin loading onto chromosomes requires the Scc2-Scc4 cohesin loader, whose presence on chromatin in budding yeast depends on the RSC chromatin remodeling complex. Here we reveal a dual role of RSC in cohesin loading. RSC acts as a chromatin receptor that recruits Scc2-Scc4 by a direct protein interaction independent of chromatin remodeling. In addition, chromatin remodeling is required to generate a nucleosome-free region that is the substrate for cohesin loading. An engineered cohesin loading module can be created by fusing the Scc2 C terminus to RSC or to other chromatin remodelers, but not to unrelated DNA binding proteins. These observations demonstrate the importance of nucleosome-free DNA for cohesin loading and provide insight into how cohesin accesses DNA during its varied chromosomal activities.  •Nucleosomes inhibit cohesin loading•Chromatin remodeling by RSC promotes cohesin loading•RSC recruits Scc2-Scc4 to chromatin independent of remodeling•Linking Scc2-C to various chromatin remodelers recreates cohesin loading Despite our increasing understanding of cohesin, how this essential protein complex accesses chromosomes is incompletely understood. Muñoz et al. provide insight into this by revealing that a molecular machine that mobilizes nucleosomes assists with cohesin loading and defines the DNA entry points for cohesin in the context of chromatin. Following DNA replication, sister chromatids are held together in a process known as sister chromatin cohesion. This is essential for faithful chromosome segregation during cell divisions. Sister chromatid cohesion is achieved by cohesin, a ring-shaped protein complex that topologically entraps both sister chromatids . The cohin vitro (in vitro but fails to load cohesin onto chromosomes in vivo. Scc4 forms a tetratricopeptide repeat superhelix that wraps around the Scc2 N terminus (Scc4-Scc2N). This module recruits the cohesin loader to chromatin in vivo  chromatin remodeling complex . RSC is Here we investigate the role of RSC in cohesin loading onto chromosomes. This reveals a dual role for this chromatin remodeler. First, RSC serves as the chromatin receptor of the cohesin loader by engaging in a direct protein interaction with the Scc2 and Scc4 subunits. This recruitment role does not require chromatin remodeling. In addition, chromatin remodeling provides nucleosome-free DNA, which is the required substrate for cohesin loading. The cohesin loading function of Scc2C can be reassigned from RSC to other chromatin remodelers but not to other DNA binding proteins. This establishes a close relationship between chromatin remodeling and cohesin loading onto chromosomes, describing the entry point by which cohesin accesses DNA in the context of chromatin.STH1 promoter with the methionine-repressible MET3 promoter, combined with Sth1 fusion to an auxin-inducible degron tag  compared with the PUG1 promoter and the tH(GUC)E1 tRNA gene. Maybe a weaker alternative Scc2-Scc4 receptor exists at ribosomal protein gene promoters. In conclusion, RSC recruits the cohesin loader independent of its ATPase, but the ATPase is required to facilitate cohesin loading.We then tested whether Scc2-Scc4 recruitment to cohesin loading sites depends on the nucleosome landscape. We measured Scc2 levels at the four cohesin loader binding sites, co-occupied by RSC, by quantitative ChIP. This analysis confirmed that cohesin loader recruitment depends on the RSC complex . Scc2 ocK501R C. Thus, in vitro assay that recapitulates cohesin loading onto DNA with purified proteins. We previously reconstituted topological DNA binding by the cohesin ring using fission yeast proteins , a single-chain nanobody with high affinity for GFP A. GBP-ScOur ability to reconstitute cohesin loading using GBP-Scc2C allowed us to investigate the requirements at chromosomal cohesin loading sites. In other words, can chromosomal proteins other than RSC serve as functional GBP-Scc2C receptors? To explore this, we fused GFP to the catalytic subunits of the other budding yeast chromatin remodelers , pioneer transcription factors , and other regulators that overlap with cohesin loading sites (Fhl1 and Hmo1) . In addiTwo additional GFP fusions allowed minimal cell proliferation in conjunction with GBP-Scc2C, those to Snf2 and to Abf1. Snf2 is the catalytic subunit of the SWI/SNF chromatin remodeler, closely related to RSC. However, Snf2 is an order of magnitude less abundant than Sth1 , which cFinally, we targeted GBP-Scc2C to the centromere using Ctf19-GFP as a receptor. This restored cohesin loading at the centromere to around half the levels seen in wild-type cells. Cohesin levels remained low along chromosomes arms E. DespitThe chromosomal loading sites of the cohesin complex at centromeres and at promoters of actively transcribed genes have been known for some time , but theOur characterization of an ATPase-deficient RSC complex revealed its role as an Scc2-Scc4 complex receptor on chromatin that is independent of RSC’s role in chromatin remodeling. Direct protein interactions exist between RSC and the cohesin loader, involving both the Scc2 and Scc4 subunits, as well as direct interactions with the cohesin complex. The RSC complex consists of 19 subunits , providiRPL19B gene promoter does not impede Scc2-Scc4 recruitment .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Frank Uhlmann . The resulting plasmids were integrated into the budding yeast genome at the TRP1 locus. Expression of wild-type Scc2, Scc2C or Scc2N was achieved by cloning the SCC2 gene under control of its own promoter into the yeast-E. coli shuttle vector pRS303, including a Pk epitope tag for detection at the C terminus. Scc2C or Scc2N fragments were derived by deleting part of the Scc2 sequence by site directed mutagenesis. The resulting plasmids were integrated into the budding yeast genome at the HIS3 locus.Epitope tagging of endogenous genes and gene deletions were performed by gene targeting using polymerase chain reaction (PCR) products. Cells were grown in rich YP medium or in complete synthetic medium (CSM) lacking methionine, supplemented with 2% glucose. To deplete Sth1 or Scc2, their gene promoters were replaced with the methionine-repressible e degron . Cells wProtein extracts for immunoblotting were prepared following cell fixation using trichloroacetic acid and separated by SDS-polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes. Antibodies used for detection are listed in the Key Resources Table and were visualized using ECL reagents and film .2). Samples were sonicated and diluted in 50 mM Tris-HCl pH 7.5. 10,000 cells per sample were analyzed using a FACSCalibur cell analyzer (BD Biosciences) and the data files were curated using FlowJo.Cells were fixed in cold 70% ethanol overnight, then treated with 0.1 mg/ml RNase A in RNase buffer (50 mM Tris-HCl pH 7.5) at 37°C for 2 hours. DNA was stained with 50 μg/ml propidium iodide in FACS buffer  using glass beads breakage in a cooled Multi-Beads Shocker (Yasui Kikai). Extracts were cleared by centrifugation, precleared and incubated with either IgG coated Dynabeads (ThermoFisher) for Protein A pulldown or with Protein A Dynabeads previously ligated to the respective epitope-specific antibody. Beads were extensively washed and elution was carried out in SDS-PAGE loading buffer.Cell extracts were prepared in EBX buffer . z stacks with 15 images at 0.1 μm intervals were acquired and merged by maximum intensity projection. Quantification of the percentage of cells showing two separated GFP foci was performed using ImageJ.Cells carrying a GFP-marked in vivo formaldehyde-crosslinked cells  and incubated at 25°C for 15 minutes. After placing on ice for 15 minutes, the binding mixtures were transferred to either antibody-coated, protein A-conjugated magnetic beads for cohesin loader and cohesin precipitation or calmodulin beads in the presence of calcium for RSC precipitation, and rocked 2 hours at 4°C. The beads were washed three times with IP buffer and once with IP buffer containing 300 mM NaCl. The bound proteins were eluted either in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, or in the presence of EGTA in the case of RSC.50 nM Scc2-Scc4, 50 nM cohesin and 50 nM RSC were mixed in 50 μL of IP buffer .Yeast histones (6.86 μg), Nap1 (14.61 μg) and ISW1A (384 ng) were mixed in a 40 μL reaction volume in buffer containing 10 mM HEPES-KOH pH 7.5, 50 mM KCl, 5 mM MgClin vitro cohesin loading assay was performed as described in  and 10% glycerol. Reactions were initiated by addition of 2.5 mM ATP and incubated for 1 hour at 29°C. Cohesin-DNA complexes were recovered by cohesin immunoprecipitation, beads were washed with high salt buffer , DNA was released from beads by proteinase K digestion and was analyzed by agarose-gel electrophoresis. Gels were stained with GelRed and bands were visualized and quantified using an Amersham Imager 600 .The ribed in , with adhttps://www.ncbi.nlm.nih.gov/geo/ with the accession number GSE117881. Unprocessed gel images presented in this manuscript can be found at https://doi.org/10.17632/34fry4t69p.1The MNase sequencing data generated in this study has been deposited with the Gene Expression Omnibus


Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination  of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the  Gene regulatory networks governing developmental processes are tightly regulated at the transcription level –5. InitiDrosophila melanogaster embryo [engrailed locus [We had previously shown that the DPE motif has an important role in the gene network that controls the development of the anterior-posterior axis of the r embryo . Nearly r embryo . Caudal r embryo –25(. Inted locus .fushi tarazu (ftz) is one of the best-characterized pair-rule genes that activates segment polarity genes. It is a homeodomain transcriptional activator that is expressed in stripes (even numbered segments) very early in anterior-posterior axis formation [ormation –30 (reviormation ). The spormation . Ftz-F1 ormation , 34 and ormation , 35–38. ormation , 39, 40.ftz promoter contains both a TATA-box and a DPE motif, and that Caudal preferentially activates the ftz promoter through the DPE motif [Drosophila species.We have previously demonstrated that the PE motif . Thus, wDrosophila embryos using the Trizol reagent (Thermo Fisher Scientific) and reverse transcribed into cDNA using MMLV-Reverse Transcriptase (Promega). The primers that were used for cloning Ftz  include a KpnI restriction site in the forward primer and a BamHI restriction site in the reverse primer. The coding region of Ftz-F1 was amplified using the Berkeley Drosophila Genome Project (BDGP) LD15303 clone as a template. The primers that were used for cloning Ftz-F1  include an EcoRI restriction site in the forward primer and a BamHI restriction site in the reverse primer. All PCR reactions were carried out using PfuUltra II polymerase (Agilent Technologies). PCR fragments were initially cloned into the pGEM-T Easy vector (Promega) before cloning into the final expression plasmids. The DNA sequence of each plasmid was verified by sequencing reactions (Hy Labs).Ftz and Ftz-F1 coding regions were cloned into the pAc5.1 ⁄ V5-His C expression vector (Thermo Fisher Scientific) without any tags. To that end, RNA was purified from 0–12 hours old CCACAATTAGG) and three Ftz-F1 DNA binding sites (TCCGAAGGACAC), based on the engrailed intronic enhancer [ftz core promoter and the firefly luciferase reporter gene of a pGL3-Basic plasmid with a modified polylinker. The DNA binding sites were assembled using three sets of annealed top and bottom oligonucleotides, each containing a Ftz-F1 DNA binding site flanked by Ftz DNA binding sites from either side . The three sets were ligated to each other and to the vector using EcoRI, XhoI, PstI and SpeI compatible restriction enzyme sites (designated by lower case letters):Each synthetic Ftz—Ftz-F1 firefly luciferase reporter plasmid contains a total of six Ftz DNA binding sites -Renilla luciferase reporter was constructed by replacing the firefly luciferase gene in the Scr-pGL3 Basic, which contains a genomic Scr fragment encompassing from −3103 to +110 relative to A+1 in the Inr [Renilla luciferase gene. The coding region of the Renilla luciferase gene was amplified by PCR with the following primers: forward AGATCTGCCACCATGACTTCGAAAGTTTATGATCC 3’5’ GA and reverse GCGGCCGCTCTAGAATTATTGTTC 3’5’  (restriction enzyme sites are underlined) using Pol III-Renilla luciferase reporter plasmid  as a template. The DNA sequence of each plasmid was verified by sequencing reactions (Hy Labs).The  the Inr , with thDrosophila Schneider S2R+ adherent cells were cultured in Schneider’s Drosophila Medium  that was supplemented with 10% heat-inactivated fetal calf serum. Cells were transfected in 24-well plates by using the Escort IV reagent (Sigma-Aldrich). For dual luciferase assays, cells were plated at 6 x 105 cells per each well of a 24-well plate one day prior to transfection. Each well was transfected with a total of 1 μg DNA composed of 930 ng of a vector control (mock -pAc empty), 60 ng of firefly luciferase Ftz and Ftz-F1 target gene reporter constructs and 2 ng of Scr-Renilla luciferase reporter. Co-activation experiments were performed by transfection of 25 ng of each expression vector . For the analyses of minimal core promoter constructs, no activator was added. Medium was replaced one day after transfection, and cells were harvested 36–48 hrs post transfection and assayed for dual luciferase activities, as specified by the manufacturer (Promega). To correct for variations in transfection efficiency, firefly luciferase activity of each sample was normalized to the corresponding Renilla luciferase activity. Each transfection was performed in triplicates, and each graph represents an average of at least 3 independent experiments.In vitro transcription reactions with Drosophila embryo nuclear extracts were carried out as previously described [Drosophila high-salt nuclear extracts [5’ TCTTCCAGCGGATAGAATGGCGCC 3’). All experiments were carried out a minimum of three independent times to ensure reproducibility of the data. Image processing and quantification of reverse transcription products were carried out using either ImageJ or Image Gauge v.3.0 (Fuji Photo Film Co.).escribed –43 usingextracts . The resDrosophila transcripts based on CAGE data [Drosophila genome) using the chi-square test. P-values were adjusted using Bonferroni correction for multiple testing.The list of Ftz target genes was composed of both previously characterized Ftz target genes , 45, 46 AGE data . Only peDrosophila melanogaster transcript were downloaded from the EPDnew database  [Drosophila melanogaster transcript were downloaded from the UCSC table browser (https://genome.ucsc.edu/cgi-bin/hgTables) “RefSeq All (ncbiRefSeq)” table, which is part of the “NCBI RefSeq” track (within the “Genes and Gene Predictions” group).The TSSs positions of each l-it.ch) , 51. Theen, drm, Sema5c, Ppa, Cad74A, noc and opa Drosophila melanogaster genes and of the corresponding exons was analyzed using the UCSC “PhyloP (phyloP27way)” table, which is part of the “Conservation” track (within the “Comparative Genomics” group). The phyloP utility is part of the PHAST  package for comparative and evolutionary genomics [Drosophila melanogaster gene. If the average conservation score of the motif sequence falls in the boundaries ±2 STD in the distribution graph, the null hypothesis is not refuted i.e., the conservation level of the area of motif sequence (downstream core promoter region) is similar to the conservation level of its related gene exons. A 35 bp sequence located 200 bp away from the core promoter region (from -241 to -275) was downloaded using the NCBI genome data viewer (https://www.ncbi.nlm.nih.gov/genome/gdv/) and used for comparison.The sequence conservation of the -2 to +33 promoter regions of the genomics , 53. Theen, drm, Sema5c, Ppa, cad74A, noc and opa) was performed using the dm6 Drosophila melanogaster genome assembly. As the TSSs of these Drosophila melanogaster genes were experimentally defined, the Multiz alignment from the UCSC genome browser (http://genome.ucsc.edu) of Drosophila melanogaster and 26 additional insect species, as well as 6 additional close gene homologs were used towards the analysis of sequence conservation, as detailed below. For every Drosophila melanogaster transcript, the position of the TSS was downloaded from EPDnew database  [Drosophila melanogaster, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, Drosophila erecta, Drosophila biarmipes, Drosophila suzukii, Drosophila ananassae, Drosophila bipectinata, Drosophila eugracilis, Drosophila elegans, Drosophila kikkawai, Drosophila takahashii, Drosophila rhopaloa, Drosophila ficusphila, Drosophila pseudoobscura, Drosophila persimilis, Drosophila miranda, Drosophila willistoni, Drosophila virilis, Drosophila mojavensis, Drosophila albomicans, Drosophila grimshawi, Musca domestica, Anopheles gambiae, Apis mellifera and Tribolium castaneum. A position weight matrix (PWM) that combines the three core promoter motifs  was constructed based on the individual PWMs of these motifs, used in the core promoter Elements Navigation Tool (ElemeNT) [en and Cad74A, the mammalian Inr (instead of the Drosophila melanogaster Inr) PWM was used, as these genes contain an Inr sequence that conforms to the mammalian Inr. For each of the sequences that were downloaded from Multiz, a program was run to detect the existence of putative elements whose PWM similarity is above a specified threshold. As listed above, most of the species’ genome sequences that were downloaded from the UCSC Multiz alignment belong to the Drosophiladea family. To check whether the combined motif also exists in gene homologs in insect families other than the Drosophiladea family, the Drosophila melanogaster proteins encoded by each of the analyzed transcripts were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). For each protein, protein blast (BLASTP) was run on non-redundant protein sequences (nr) database excluding the Drosophilidae family (taxon identifier 7214). From the list of results of each one of the BLASTP queries, the top six species with highest coverage & identity were chosen. The gene coordinates for each of these species were downloaded from NCBI. Since there was no available information on the transcription start sites in these species, the sequence of ±1000 bp around the annotated gene start position was downloaded from NCBI. Similarly to the process run for the sequences downloaded from UCSC Multiz alignment, using a PWM that combines the three core promoter motifs , a program was run to detect the existence of putative elements whose PWM similarity is above a specified threshold on every one of the downloaded sequences. Motif sequences with PWM scores higher than 5 that were located within ±200 bp from the TSS or the annotated gene start positions were chosen. With these defined thresholds, there was only one motif detected for most species. In case more than one motif was detected per species, the motif that was closer to the TSS or gene start was chosen. The motif logos were generated using WebLogo (https://weblogo.berkeley.edu/) [Evolutionary conservation analysis of the Inr, BridgeI and DPE combination within the core promoters of seven Ftz target genes (l-it.ch) , 51. BasElemeNT) . For en ey.edu/) , 55.ftz [ftz gene contains a TATA box, an Inr and a DPE motif [ftz promoter with a distinct preference for the DPE motif over the TATA box [Caudal is a homeodomain sequence-specific DNA binding transcription factor that has been shown to bind and activate the transcription of ftz . We had PE motif . MoreoveTATA box , 57. We It has been shown that the specificity of Ftz activity is achieved by its interaction with its obligatory co-factor, the orphan nuclear receptor Ftz-F1 , 37, 58.engrailed enhancer [ftz DPE-dependent core promoter  or a ftz TATA-dependent core promoter   . ImportadENCODE, ), were cNeither Ftz nor the Ftz-F1 co-factor alone significantly activated the TATA-dependent (mDPE) or the DPE-dependent (mTATA) synthetic reporter . The synDrosophila melanogaster CORE database and the Elements Navigation Tool (ElemeNT) [Drosophila melanogaster TSSs [Drosophila genome  , which ater TSSs  and anala genome . By defiDrosophila melanogaster Ftz target genes. The analyzed genes included the well-characterized en [drm, Sema5c, and noc Ftz target genes identified in Ftz-F1 mutants [Ppa, Cad74A and opa genes [opa, which only contains an Inr and DPE. Interestingly, en and Cad74A sequences conform to the mammalian initiator sequence, while the other promoter sequences match both Drosophila and mammalian initiator motifs. All of the examined genes are co-expressed with Ftz and Ftz-F1 during early embryonic stages of development We first calculated the distribution of the average conservation scores of 1000 randomly chosen 35 bp sequences within exons of each of the abovementioned genes. Since the coding region is known to be highly conserved, the calculated conservation scores were used as a reference for the conservation level of the -2 to +33 region of the corresponding gene. The conservation analysis was based on the phyloP utility within the PHAST  package for comparative and evolutionary genomics , 53. Theen, drm and opa, but not for Sema5c, Ppa, Cad74A and noc. Notably, the conservation levels of the core promoter regions of all analyzed Ftz target genes were higher than the conservation levels of the upstream control regions, further implicating the conserved downstream core promoter region in the regulation of these genes.We then used a 35 bp sequence located 200 bp upstream of the core promoter region (from -241 to -275) as a control. This upstream sequence was conserved for Drosophila melanogaster Ftz target genes using the Multiz alignment from the UCSC genome browser (http://genome.ucsc.edu), which includes Drosophila melanogaster, 26 additional insect species, and 6 additional close gene homologs  for the seven Ftz target genes analyzed above. In order to distinguish between the effect of the DPE and/or the Bridge motif on the reporter activity, we have generated four distinct core promoter versions for each gene—WT, mutant DPE (mDPE), mDPE only (27–29) and mBridgeI (18–22)  . The locCad74A and noc luciferase reporter genes, as compared to their effects on the activities of the luciferase reporters driven by en, drm, Sema5c and Ppa. In addition, the effect of mBridgeI was more pronounced for en, drm, Sema5c and Ppa, and less pronounced for Cad74A and noc reporter genes. As expected, the mBridgeI had no effect on the activity of opa, which lacks a BridgeI motif. Taken together, these results emphasize the contribution of the downstream core promoter nucleotide composition to the transcriptional activity.Mutations in the DPE sequence of each of the seven target genes reduced the activities of the luciferase reporter by more than 2-fold . Partialin vitro transcription using Drosophila embryo nuclear extracts, followed by primer extension analysis  proteins , preferentially activate transcription of the ftz promoters via the DPE core promoter motif [Renilla luciferase as normalization controls. This was likely due to the fact that genomic ftz reporters receive input from multiple cellular regulators. Thus, we have used a unique experimental setting utilizing reporter plasmids containing synthetic Ftz and Ftz-F1 binding sites. It remains to be determined whether CBP, which has been shown to contribute to DPE-preferential activation [The entation . Caudal thropods , 63. Herer motif , 57, andDrosophila genome. We thoroughly analyzed the promoter regions of seven Drosophila melanogaster Ftz target genes: the well-characterized en [drm, Sema5c, and noc Ftz target genes that were identified in Ftz-F1 mutants [Ppa, Cad74A and opa genes [en, drm, Sema5c, Ppa, cad74A and noc) contain functional Inr, BridgeI and DPE motifs. Interestingly, this further highlights the similarity between the well characterized and the less characterized Ftz target genes.We observed an enrichment of genes that contain a combination of Inr, Bridge and DPE motifs in Ftz target genes, as compared to the core promoter composition of the rized en , 35, 38, mutants  and the pa genes , 60. We in vitro transcription analysis using embryo extracts, the transcriptional output is mostly dependent on the DPE motif, with different downstream regions, including the BridgeI motif, contributing to it. This is in line with a sequence bias revealed in position +19 in DPE promoters [As demonstrated by both reporter assays in cell culture and romoters .The DPE motif was previously shown to be important for the regulation of the Hox genes, as well as for the dorsal-ventral gene regulatory network , 64. HerIn general, these results further highlight the importance of the downstream core promoter region for transcriptional regulation of Ftz target genes, among other developmental programs. We demonstrated that BridgeI is an “auxiliary” element, which supports the function of DPE-dependent transcription in the examined promoters, but is not sufficient for fully restoring the transcriptional activity upon DPE loss. This is also supported by the fact that most examined functional DPE motifs are accompanied by the Bridge motif, while non-functional ones usually lack it. Potentially, this could promote robustness, where a strong DPE accompanied by a Bridge motif ensures proper docking of TFIID for accurate transcriptional activity.Drosophila species. Interestingly, based on this analysis, most of the identified motifs in non-Drosophiladea belong to the Diptera order. In fact, the only gene with orthologs outside Diptera is drm, which is highly conserved and has homologs in orders that include beetles (Coleoptera), butterflies and moths (Lepidoptera). Although the Ftz-F1 co-factor is conserved to bilaterians, no non-Dipteran Ftz orthologs have been identified (Flybase.org). This may explain why the specific core promoter combination is conserved to Diptera and additional insects, but not to higher multicellular organisms. This is another indication that it is Ftz, rather than Ftz-F1, that has the ability to activate with a preference for the specific core promoter motif. This adds another layer of complexity to this complex developmental transcriptional network. Taken together, we identified functional evolutionarily conserved downstream core promoter elements that are important for the transcriptional regulation of Ftz target genes.The conservation of the combination of Inr, BridgeI and DPE motifs, as well as the strict spacing between them, may suggest the functionality of these regulatory sequences in other S1 Fighttps://prosite.expasy.org/cgi-bin/prosite/mydomains/).Ftz and Caudal protein sequences, with Homeobox domain highlighted in yellow. Additional similarities, as detected by BLASTP, are indicated. Each unique color indicates a different stretch of similarity. Graphical representation of the similarity stretches was generated using MyDomains—Image Creator Click here for additional data file.S2 Figftz, ftz-f1 (orange color tones) and the examined Ftz targets (cyan color tones) were based on modENCODE data [Expression levels of ODE data , as pres(PDF)Click here for additional data file.


Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available experimental data on HMGA1 and HMGA2 functions in governing embryonic and adult stem cell fate. Moreover, based on the available evidence, we will aim to outline how HMGA expression is regulated in different contexts and how these two proteins contribute to the regulation of gene expression and chromatin architecture in stem cells.HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed.  HMGA1 and HMGA2 orthologues are present in mammals, birds, fishes and reptiles. HMGA1 orthologues have been found in 254 species, while 231 organisms have a HMGA2 ortholog (NCBI Gene data bank). Similar proteins are also present in many taxa, including plants, where it is difficult to assign the homology to HMGA1 or HMGA2, due to the high similarity of these two proteins, which probably evolved from a single ancestor. HMGA1 and HMGA2 are small proteins with a very similar structure that includes three conserved domains, also known as AT-hooks, and a C-terminal domain rich in acidic residues. Each AT-hook domain contains a characteristic stretch of 9 residues, mostly Arg and Lys, which interacts with a stretch of 15 AT bp with high affinity [High mobility group A (HMGA) proteins are non-histone chromatin proteins. Their classification as a high mobility group refers to their rapid electrophoretic migration, in part due to their small sizes 10–15 kDa). The analysis of available nucleotide sequences shows that 0–15 kDa.Hmga1 is expressed until 16.5 dpc (days post coitum), whereas Hmga2 disappears already at 14.5 dpc [Hmga1 expression was detectable in the yolk sacs of wild-type mice at 9.5 dpc. Its expression decreases at 14.5 dpc and increases in the fetal liver [Hmga2 in the somite, tail and eye at 9.5 dpc with a localization confined only in the eye at 13.5 dpc (MGI database). In 14.5 dpc mouse embryos, Hmga1 is highly expressed in several tissues such as the epidermis, stomach, midgut and hindgut, testis, lung, pancreas, thyroid and thymus primordium . In later phases of fetal development and in most adult tissues, these two proteins are not detectable [Hmga2 was found in preadipocytic proliferating cells, spermatids, and spermatocytes [HMGA proteins are highly expressed during embryonic development as demonstrated by Northern blot and in situ hybridization analyses on mouse embryos, showing that 14.5 dpc . Hmga1 eal liver . The avatectable . Only HmHMGA2 gene were found [HMGA1 gene rearrangements in lipomas, uterine leiomyomas, pulmonary chondroid hamartomas [Hmga2 in the tumorigenesis was supported by the spontaneous development of lipomas and of other types of benign tumors in mice overexpressing Hmga2 or its truncated forms [On the other hand, HMGA proteins are often expressed at very high levels in cancers . These ire found . The latmartomas ,10. The ed forms ,12,13.HMGA1 or HMGA2 have been frequently found [HMGA1 and HMGA2. Despite the large number of studies focusing on HMGA proteins, their functions in normal cells are not definitively understood. The main hurdle in addressing this issue is probably that most of the experimental data that could contribute to the understanding of HMGA proteins was obtained in tumor cells. Indeed, in many cases, what is observed in cancer cells does not necessarily take place, exactly in the same fashion, in a normal cell. For example, in the case of HMGA proteins, it was demonstrated that they accumulate in senescent cells and that their knockdown by RNAi resulted in a partial bypass of the senescence induced by oncogenic Ras [In malignant cancers, elevated levels of either ly found ,15. Simienic Ras . These dWe find that the multiple roles of HMGA proteins in physiological contexts are intriguing and still not completely understood. Thus, considering that HMGA proteins in cancer are already extensively described and reviewed ,17,18, tHmga1 and Hmga2 knockout mice clearly indicate their crucial role in the different phases of mouse development. Hmga1 KO mice show a complex phenotype including the downregulation of insulin receptors and consequent glucose intolerance and cardiac hypertrophy [Hmga1−/− yolk sacs and in 14.5-dpc fetal livers compared with wild-type tissues [Hmga1 in regulating hematopoiesis, in agreement with data obtained in ESCs (see below). Hmga2 gene KO in mice is responsible for a specific phenotype, also known as pigmy, consisting in reduced weight at birth, shortened head, and a body weight of adult mice that is about 40% of the normal weight [Hmga2 KO mice [Hmga1 and Hmga2 probably partially compensate for the absence of each other, as suggested by the dramatic phenotype of the Hmga1/Hmga2 double KO mice, which show a 70% reduction of the body weight at birth, which is not compatible with survival [Hmga1/Hmga2-null mice also showed embryonic lethality, indicating that the HMGA proteins play a critical role during embryonic and/or fetal development [The phenotypes observed in ertrophy ,20. In t tissues . This sul weight . Further KO mice . Hmga1 asurvival . Intereselopment .Hmga2 show a characteristic phenotype, including overgrowth with an enlargement of the adipose tissue [Hmga2 roles in humans, where its chromosomal rearrangement, leading to mRNA truncation, was described in a patient showing dramatic somatic overgrowth [Mice overexpressing e tissue . This phergrowth .Hmga1 is highly expressed in mouse ESCs (mESCs) and decreases during differentiation, playing a crucial role in their lymphohematopoietic differentiation. Indeed, Hmga1 KO ESCs showed a decrease in the formation of T-cell precursors balanced by an increase in B cell generation. Moreover, the absence of Hmga1 expression induces a reduction in monocyte/macrophage population and an increase in megakaryocyte precursor numbers, erythropoiesis, and globin gene expression [HMGA1 is also expressed in undifferentiated human ESCs (hESCs) and its levels decline during differentiation [HMGA1 blocks the differentiation of hESCs, maintaining the undifferentiated state, as demonstrated by the resulting high levels of stemness markers such as OCT4, NANOG, SOX2 and MYC. Accordingly, the suppression of HMGA1 by RNA interference in hESCs resulted in a downregulation of pluripotency genes, SOX2, OCT4, cMYC and LIN28, likely by direct interaction of HMGA1 with their promoters, at least SOX2, MYC and LIN28 [HMGA1 also shows a role in enhancing the establishment of a pluripotent stem cell phenotype through cell reprogramming. In 2006, Takahashi and Yamanaka demonstrated that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by four transcription factors (TFs): OCT4, SOX2, KLF4, c-MYC (OSKM), also called the Yamanaka cocktail [HMGA1 led to a two-fold increase in human mesenchymal stem cell (MSC) reprogramming efficiency when added to the Yamanaka cocktail. This effect is due to the HMGA1-dependent expression of a subset of pluripotency- associated genes during the early phases of the reprogramming process [In physiological contexts, pression . HMGA1 intiation . This exnd LIN28 . In addicocktail . These ccocktail . Among t process .HMGA2 in hESCs are consistent with a role of HMGA proteins in controlling stem cell identity and differentiation through regulation of gene expression in these cells. Indeed, HMGA2 is expressed at high levels in hESCs and is further up-regulated during a short time window in the very early stages of hESC differentiation [HMGA2 knockdown showing that HMGA2 regulates the expression of genes linked to self-renewal and proliferation as well as mesodermal differentiation in hESCs [HMGA2 in hESCs in addition to that of gene expression regulation. Yu and colleagues demonstrated that HMGA2 is required to protect stalled replication forks against nucleolytic collapse in hESCs as well as cancer cells that, exhibiting fast DNA replication cycles, frequently encounter stalling of replication forks [The data reported for ntiation . In thesin hESCs . A more on forks .Hmga2 is barely detectable but it promptly accumulates upon the induction of differentiation [Hmga2 in EpiLCs is in agreement with the expression in hESCs that actually behave as mouse epiblast stem cells. Hmga2 induction during the early phases of mESC differentiation is required for the exit from the naïve state. Indeed, the suppression of Hmga2, by either gene KO or silencing, blocks the differentiation program. On the other hand, persistence of high levels of Hmga2 during differentiation perturbs the cell cycle and increases the apoptosis rate of differentiating cells [Hmga2 expression must be tightly controlled to allow the proper ESC differentiation. The mechanisms through which Hmga2 affects mESC differentiation involve the HMGA2-dependent regulation of differentiation genes. Upon the exit from the undifferentiated state, mESCs acquire a phenotype resembling that of the epiblast stem cells that are primed for further differentiation [Hmga2 itself is one of these genes, thus a feedforward loop based on the induction of Hmga2 expression sustains the change in the expression profile of differentiating mESCs.In mESCs, ntiation ,30 into ng cells , thus inntiation . This phntiation ,33. ChIPntiation . Hmga2 iHmga2 KO mice [Hmga2 in mESCs specifically favors myogenic differentiation without affecting other cell lineages [In agreement with the described role of HMGA2 in allowing the differentiation of mESCs  and the  KO mice , the ovelineages .HMGA2 compared to dermal fibroblasts, are more prone to be reprogrammed into iNSCs [Hmga2 showed a strong decrease in reprogramming efficiency, although some reprogrammed colonies can be obtained [As observed in the case of HMGA1, HMGA2 can promote adult cell reprogramming toward a stem cell phenotype. Human dermal fibroblasts or mouse embryonic fibroblasts (MEFs) can be directly converted into induced neural stem cells (iNSCs) by simply transducing them with the transcription factor SOX2 . The co-to iNSCs . Thus, hobtained .Hmga1 is enriched in intestinal stem cells (ISCs). In conditional transgenic mice, Hmga1 overexpression amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. HMGA1 also helps to “build” an ISC niche by expanding the Paneth cell compartment. Moreover, HMGA1 resulted in the organization of ISCs into three-dimensional organoids in vitro. These experimental data indicated a role for HMGA1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche [ll niche ,37.Hmga2 has been detected in NSCs and progenitor cells of the subventricular zone of newborn mice [Hmga2 present in these cells declines with age, becoming undetectable in old mice. Experiments in Hmga2 KO mice demonstrated that this protein is necessary for the self-renewal of NSCs. In addition, this effect could be based on HMGA2-mediated regulation of gene expression. Indeed, HMGA2 sustains self-renewal of NSCs by downregulating p16Ink4a and p19arf, and ChIP experiments have indicated a direct interaction of HMGA2 with the JunB gene, which in turn regulates p16Ink4a and p19arf [Hmga1 does not decrease with age, thus appearing not to be involved in this regulatory mechanism. However, these data are not completely in agreement with the observations of Kishi and co-workers [Hmga1 and/or Hmga2 affect chromatin condensation in NPCs of mouse neocortex, and the decline of Hmga expression parallels the increasing condensation of the chromatin. In agreement with ChIP-seq data upon overexpression of Hmga in mESCs [Hmga ectopic expression has a more general effect on chromatin structure, instead of an effect targeted to specific gene loci.In adult stem cells, orn mice . The amod p19arf . It is w-workers  who, thrin mESCs , these dHmga2 in neurogenesis is also supported by further evidence. Hmga2 is expressed at a high level in the neocortex at E12.5, but rapidly declines, becoming undetectable at E15.5. The overexpression of Hmga2 causes a rearrangement of the neocortical layers, with the Hmga2 overexpressing cells retained in the profound layers, while the in utero silencing of Hmga2 has an opposite effect, shifting the Hmga2 knockdown cells towards more superficial layers [Hmga2 KO mice, the number of cells present in the gut enteric nervous system that form neurospheres at E14.5 is similar to the number found in the wild type gut, but this number significantly declines at P0 and in the adult mice. In all cases, the size of the neurospheres is always reduced in the Hmga2 KO mice [The role of l layers . Moreove KO mice .Hmga2 are associated with an expansion of the hematopoietic compartment, including HSCs [Hmga2 KO mouse embryos were transplanted in irradiated congenic mice, the frequency of donor-derived HSCs is reduced compared to that observed when wild type cells were transplanted, thus indicating a decreased self-renewal capacity of Hmga2 KO HSCs [Hmga2 KO HSCs suggested that the Igf2bp2 gene could be directly regulated by HMGA2 and, at least in part, responsible for the observed phenotype. The reduced size of skeletal muscle found in Hmga2 KO mice (see above) supports the idea that HMGA2 can directly affect Igf2bp2 gene expression by regulating its transcription. Indeed, Igf2bp2 mRNA and protein are decreased in Hmga2 KO myoblasts and, on the contrary, Hmga2 overexpression in these cells results in the accumulation of Igf2bp2 mRNA and protein. IGF2BP2 seems to be an important player downstream of HMGA2, as its ectopic expression partially reverts the Hmga2 KO muscle phenotype [In the fetal hematopoietic compartment, HMGA2 is necessary to sustain the self-renewal capacity of mouse hematopoietic stem cells (HSCs). Ikeda and coworkers first observed that high levels of ing HSCs . When HS KO HSCs . The mechenotype .HMGA2 was detected for the first time in CD34-positive cells [In human HSCs, ve cells , and appve cells .HMGA2 contribution to human HSC differentiation showed that the suppression of HMGA2 leads to a decrease of myeloid progenitor cells but has no effects on the differentiation of these cells. On the contrary, HMGA2 is necessary for both erythroid precursor propagation and differentiation [β-globin gene transfer in an adult patient with severe βE/β0-thalassaemia was correlated to the transcriptional activation of HMGA2 mRNA in erythroid stem/progenitor cells, which is accompanied by a benign cell expansion [A detailed analysis of the ntiation . Of notexpansion .HMGA2 overexpression blocks the differentiation of hMSCs into the osteogenic lineage by limiting the expression of the osteogenic factor RUNX2 and, conversely, the down-regulation of endogenous HMGA2 promotes osteogenic differentiation [The role of HMGA2 in sustaining self-renewal of adult stem cells possibly by blocking their differentiation was also described in hMSCs. Indeed, ntiation ,48,49.HMGA2 expression, in addition to its effect in promoting osteogenic differentiation, also leads to a severe impairment of adipogenesis [Hmga2 KO mice that shows a striking reduction in adipose tissue, and with transgenic mice overexpressing Hmga2 showing somatic overgrowth and, in particular, increased abundance of fat and lipomas [Interestingly, HMGA2 may have a dual role in differentiation of adult stem cells. Indeed, in hMSCs the silencing of ogenesis . This roogenesis . These o lipomas ,23.HMGA1 is highly expressed in human colon tumor stem cell lines and its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency, and recovers the expression of NUMB, an endocytic protein promoting asymmetric division that is typical of normal stem cells [HMGA1 is highly expressed and upon its silencing the CD133+/CD15+ stem cell population is reduced [NUMB both at transcriptional level and post-transcriptionally through the regulation of the RNA binding protein MSI1 and the miR-146a expression [All these experimental data point to a general role of HMGA proteins in regulating the balance between self-renewal and differentiation of stem cells . This isem cells . The sam reduced . In thespression .IFN-β gene promoter. Upon viral infection, the transcription of the IFN-β gene depends on the recruitment of several TFs, including NFkB, to an enhancer element within the IFN-β gene promoter. The assembly of this complex is dependent on the interaction of HMGA1 with an A/T-rich sequence present in the promoter [IL-2Rα gene. Hmga1 is upregulated upon stimulation of T cells and binds to A/T-rich sequences in the IL-2Rα gene promoter inducing a chromatin remodeling that allows the accessibility of regulatory cis-elements to several TFs, like ELF-1, STAT5 and others [IL-2Rα and α-B-crystallin gene promoters [Many of the experimental data reported above suggest that HMGA proteins contribute to regulating chromatin structure, and thus influencing gene expression. A direct binding to chromatin of HMGA1 or HMGA2 was described in several different conditions. These proteins preferentially bind to A/T-rich sequences near, or overlapping with, binding sites of TFs orchestrating the assembly of multi-subunit protein–DNA complexes (enhanceosomes) by modifying the chromatin structure in an ATP-independent fashion . One exapromoter . Anotherd others . Duncan romoters . In the romoters . In hESCromoters ,27. Furtromoters .Brca1 promoter in mESCs, repressing its expression [Hmga1 KO and wt mESCs have identified 87 transcripts increased and 163 decreased in the absence of HMGA1. Many of these target genes showed cell- and tissue-specific regulation by HMGA1 when compared with the results obtained in MEFs, liver, spleen and heart from wt and Hmga1 KO mice [Hmga1 or Hmga2 in undifferentiated mESCs results in a diffuse binding of these proteins to chromatin mainly at heterochromatic regions [One of the effects of HMGA seems to be that of removing the nucleosomal constraints that prevent the formation of the TF-DNA complexes. This effect can be explained by the observation that the chromatin binding sites of the HMGA proteins are like those of histone H1 indicating that HMGA proteins compete with H1 for binding to linker DNA, thereby inducing a loosening of the chromatin structure ,59. Howepression . Microar regions . This diLin-14 mRNA through an RNA–RNA interaction [C. elegans for its ability to regulate expression of multiple mRNA targets [Hmga2 mRNA is one of the targets of let-7. Thus, as let-7 induces the downregulation of HMGA2 protein, the expression profile of these two molecules is specular: HMGA2 is expressed in undifferentiated cells, while let-7 miRNAs are expressed in differentiated cells. A further key player in this regulatory mechanism is LIN28. LIN28A and B are two RNA binding proteins that are able to limit the biogenesis of a subset of miRNAs, and in particular those of the let-7 family. The main mechanisms underlying this regulation are based on the direct interaction of LIN28B with the pri-let-7 RNA, preventing the processing of this molecule by the microprocessor complex in the nucleus [An important advancement in the understanding of HMGA2 functions emerged from the discovery of a complex crosstalk among this protein and other molecules involved in several differentiation programs. Following the discovery of the small RNA lin-4 in 2000, which is able to regulate the translation of the eraction , the mic targets . In mamm nucleus  and of L nucleus ,66. Take nucleus ,38,43).Hmga2 and Lin28 maintain the stemness of mouse RPCs. During late retinal histogenesis, let-7 increases and negatively regulates the translation of Hmga2/Lin28. Low levels of Hmga2/Lin28 and high levels of let-7 in RPCs shift the balance from RPC maintenance to their differentiation [A beautiful example of the effects of the modulation of the LIN28-let7-HMGA2 axis is represented by retinal progenitor cells (RPCs), where let-7 facilitates differentiation. In this context, high levels of ntiation .Lin28 in myeloid progenitors parallels the accumulation of mature let-7. The inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28 or ectopic activation of let-7 in the fetal state induces a shift toward the adult-like myeloerythroid phenotype. Furthermore, the analysis of the effects induced by Hmga2 ectopic expression indicated that this architectural factor as an effector of LIN28-let-7-controlled myeloerythropoiesis [The LIN28-let7-HMGA2 axis also fulfills a crucial role in hematopoietic maturation to adulthood in mice. The decrease of opoiesis .HMGA2 through direct targeting [LIN28 expression in MSCs reduces the expression of let-7 and up-regulates that of HMGA2, which in turn activates the transcription of pluripotency-associated factors, maintaining the stem cell phenotype [In human adipose tissue-derived MSCs, let-7 positively regulates osteogenic differentiation by repressing argeting . Accordihenotype .HMGA2 can be controlled by other miRNAs that might synergize with let-7 in a context-dependent manner [HMGA2 repression by the microRNA (miR)-664a-5p is required for proper osteogenic differentiation. Consistently, in these cells, overexpression of HMGA2 counteracts the stimulatory effect of miR-664a-5p on osteogenic differentiation [HMGA2 mRNA is required to properly fulfill the differentiation program [HMGA2 itself and LIN28 mRNAs whose translation is favored by IGF2BP2 [HMGA2 gene. At the C/G skew present in the HMGA2 gene promoter, the ncRNA forms an R-loop with the genomic DNA, thus favoring chromatin decompaction and the transcription of the HMGA2 gene [IGF2BP2 promotes the binding of IGF2BP2 to the HMGA2 mRNA, thus favoring its translation. It is worth noting that RPSAP52 does not affect the binding of IGF2BP2 to other known mRNA targets, like HMGA1, NRAS and IGF1R. RPSAP52 may also act through an additional competing endogenous RNA (ceRNA)-based mechanism, as it can titrate several miRNAs targeting HMGA1 and HMGA2, thus leading to their increased expression [Other recent reports have also shown that in hMSCs, the expression of t manner ,48,49. I program ,70. Amon IGF2BP2 ,72,73. Tpression . These oHmga2 expression through the cooperation with OTX2, Hmga2 expression is regulated by LIN28 in a let-7-independent manner [Hmga2 mRNA, and this negatively controls its translation. This mechanism prevents the inappropriate accumulation of HMGA2 during differentiation that would modify the proliferation and physiological apoptosis of ESCs. On the other hand, upon the exit of mESCs from the undifferentiated state and the transition into epiblast-like cell states, LIN28 accumulates along with HMGA2. This induction of Lin28 is dependent on the direct interaction of OTX2 and HMGA2 with Lin28 genes [In mESCs, although many miRNAs have a fundamental role in controlling gene expression, targeting specific mRNAs and regulating the exit from the pluripotent state ,78,79,80t manner . Indeed,28 genes .HMGA2 gene and, together with IGF2BP2, activates the translation of the HMGA2 mRNA. HMGA2 levels are negatively regulated by let-7 miRNAs. In addition, IGF2BP2 could regulate let-7 levels by masking miRNA target sites on various mRNAs, thus favoring let-7 degradation. This is expected to result in a further increase of HMGA2 biosynthesis. In addition, both HMGA2 and IGF2BP2, through independent mechanisms, favor the expression of LIN28 and this in turn contributes to the downregulation of let-7 miRNAs. Therefore, there are several positive feedback loops, all contributing to increase the amount of HMGA2. Two questions remain to be addressed. First, in cells expressing HMGA2, LIN28 and IGF2BP2, a negative regulator is necessary to balance the positive loops described above. In mESCs differentiating into EpiLCs, where let-7 miRNAs do not accumulate, this indispensable negative regulator of Hmga2 could be LIN28, which hampers the translation of Hmga2 mRNA. Second, in differentiating adult stem cells, the exit from the “undifferentiated” state could be triggered by the inhibition of Lin28 expression that results in accumulation of let-7 miRNAs. Furthermore, other factors can contribute to downregulation of HMGA2 levels in cell-specific contexts, such as other specific miRNAs possibly synergizing with let-7 [Several reports ,70,71,73th let-7 ,48,49, ath let-7  or the sth let-7 .Hmga1 expression, which in turn leads to the increased expression of myogenic differentiation genes to allow myotube formation [Hes5 in mice inhibited neuronal differentiation from NSCs, while gliogenesis was also accelerated and enhanced. In these mice, Hmga1 and Hmga2 expression was suppressed in the neocortical regions. By contrast, in the Hes5 knockout (KO) mice, the transition of neurogenesis and gliogenesis was delayed and this effect was accompanied by the upregulation of Hmga1 and Hmga2. In utero electroporation of shRNA targeting Hmga1 and Hmga2 in Hes5 KO mice showed that the transition timing of layer-specific neurogenesis and astrogenesis was regulated at least in part by the expression levels of Hmga1 and Hmga2 genes [Hmga1 and Hmga2 KO MEFs suggested the existence of a feedback loop among these two factors that reciprocally regulates each other. Indeed, in Hmga1 KO MEFs, the expression of HMGA2 protein is increased and in Hmga2 KO MEFs the expression of HMGA1 protein is increased [Hmga1 and Hmga2 did not show significant differences in KO MEF lines [Hmga1 expression through specific miRNAs is well described in different cell contexts [HMGA1 is high and is inversely correlated with miR-214 expression. Luciferase assays and western blot demonstrated that miR-214 overexpression reduces the level of HMGA1 and counteracts its effects on proliferation, migration and invasion in cervical and colorectal cells [Hmga1 expression is inversely correlated with the expression of miR-195/497. The abundance of the HMGA1 protein was reduced in C2C12 cells after the induction of the myogenic program, which is opposite to the upregulated expression of the miR-195/497. The Hmga1 3’ UTR luciferase reporter assays in C2C12 cells showed that the ectopic miR-195/497 significantly repressed the luciferase activity. Consistent with this data, ectopic expression of miR-195/497 reduced the level of HMGA1 in C2C12 cells [HMGA1 is highly expressed. In these cells, the transfection of let-7i mimics reduced HMGA1 mRNA and protein expression suggesting that HMGA1 is a target for let-7i [HMGA1 mRNA 3′UTR and inhibiting its degradation [On the other side, little experimental evidence is available on the regulation of HMGA1 expression. In the mouse myoblast cell line, C2C12, a PKCε–HMGA1 signaling axis was described that regulates skeletal muscle differentiation. In these cells, the kinase PKCε down-regulates ormation . The wora2 genes . Interesncreased . This efEF lines . A post-contexts ,84,85. Ial cells . During radation . Thus, fHMGA2 is highly expressed in hESCs [Hmga2 starts to be expressed [The data discussed in this review point to a crucial role of HMGA proteins in the control of the balance between self-renewal and differentiation in embryonic and adult stem cells . These din hESCs ,28, wherin hESCs ,30, and xpressed ,47,48,49xpressed ,47,48,49Hmga1 and Hmga2 leads to an increase in reprogramming efficiency. Cell reprogramming mechanisms are mostly based on the epigenetic remodeling of the starting cells to allow the profound changes in chromatin organization and gene expression needed to acquire the reprogrammed phenotype. Most factors that are able to improve reprogramming efficiency, called reprogramming enhancers, are epigenetic modulators. In this context, HMGA proteins can be considered reprogramming enhancers and their role in this mechanism is possibly correlated with their ability to change chromatin organization at different levels. Indeed, the binding affinity of HMGA proteins for A/T rich sequences allows one to recognize countless points on the genome and thus intragenic or intergenic regulatory elements (enhancers and silencers), as well as gene empty regions. In this way, HMGA proteins may have a double role in changing the epigenetic profile. At the local level, they allow the accessibility to pioneer TFs on naïve chromatin to allow the entry of canonical TFs, probably by displacing H1 histone and removing chromatin constrains that block the assembly of TF-DNA complexes (For this reason, the HMGA effects on cell reprogramming are very intriguing. HMGA2 is required to efficiently obtain mouse iPSCs and the overexpression of both omplexes A. At theomplexes B. Both t