IBD5 locus (human 5q31), that harbors the IRF1 gene. A cause-to-effect relationship between chronic inflammation and colorectal cancer, and a possible role of IRF1 were studied in Irf1-/- mice in a model of colitis-associated colorectal cancer (CA-CRC) induced by azoxymethane and dextran sulfate. Loss of Irf1 causes hyper-susceptibility to CA-CRC, with early onset and increased number of tumors leading to rapid lethality. Transcript profiling (RNA-seq) and immunostaining of colons shows heightened inflammation and enhanced enterocyte proliferation in Irf1−/− mutants, prior to appearance of tumors. Considerable infiltration of leukocytes is seen in Irf1−/− colons at this early stage, and is composed primarily of proinflammatory Gr1+ Cd11b+ myeloid cells and other granulocytes, as well as CD4+ lymphoid cells. Differential susceptibility to CA-CRC of Irf1−/− vs. B6 controls is fully transferable through hematopoietic cells as observed in bone marrow chimera studies. Transcript signatures seen in Irf1−/− mice in response to AOM/DSS are enriched in clinical specimens from patients with IBD and with colorectal cancer. In addition, IRF1 expression in the colon is significantly decreased in late stage colorectal cancer  and is associated with poorer prognosis. This suggests that partial or complete loss of IRF1 expression alters the type, number, and function of immune cells in situ during chronic inflammation, possibly via the creation of a tumor-promoting environment.The mechanisms linking chronic inflammation of the gut (IBD) and increased colorectal cancer susceptibility are poorly understood. IBD risk is influenced by genetic factors, including the  The etiology of colorectal cancer involves both a strong genetic contribution and complex environmental and socioeconomic determinants including diet, lifestyle, and smoking3. A positive risk factor for colorectal cancer is a prior history of chronic intestinal inflammation. Indeed, patients with inflammatory bowel disease (IBD) are at greater risk of developing colorectal cancer than the general population4. IBD represents intestinal disorders that comprise Crohn’s disease (CD) and ulcerative colitis (UC) resulting in lifelong chronic inflammation of different segments of the intestine5. Colitis-associated colorectal cancer (CA-CRC) is the most severe consequence of IBD.Colorectal cancer was the third most commonly diagnosed cancer worldwide in 20188. However, the specific contributions of these loci, individually or as a group, to CA-CRC risk remain unknown. Such genetic loci may encode proteins of significant value as potential drug targets, and as predictive markers to identify IBD patient subsets with increased risk of developing CA-CRC. For instance, the IBD5 risk locus (5q31-q33) was first associated to CD by genome scan in 158 Canadian sib-pair families with segregating disease9. The IBD5 association was replicated and refined by GWAS to a region that maps to a segment of human Chromosome 5 (5q31.1) containing ~20 genes in linkage disequilibrium with a number of attractive positional candidates, including the transcription factor Interferon regulatory factor 1 (IRF1)7. Although multiple genes at the IBD5 locus were studied for their putative causality under the associated haplotype10, to date, none have been functionally validated.Population studies have demonstrated that in humans, IBD is under complex genetic control with >225 risk loci mapped in large cohort association studies (GWAS)11. Upon induction, IRF1 stimulates transcription of IFN-inducible genes involved in innate and acquired immunity. Irf1-deficient mice display a severe immunodeficiency, with alterations in myeloid cell maturation, CD8+ T cell numbers, and impaired NK cell cytolytic activity15. Irf1−/− mice show active colitis in response to the irritant dextran sodium sulfate (DSS)17, and human IRF1 mRNA is increased in lamina propria mononuclear cells from CD patients18, thus suggesting that IRF1 may contribute to inflammation in IBD, and possibly to progression to CA-CRC.IRF1 is a transcription factor that plays a key role in the development and function of myeloid and lymphoid cells. IRF1 is expressed constitutively at low levels in many cell types and is induced in response to various stimuli, including viral and bacterial infections, and cytokines such as IFNγ and TNFαIrf1−/− mice display increased tumorigenesis in response to azoxymethane and dextran sodium sulfate (AOM/DSS)-induced CA-CRC. Combination of immune cell profiling and comparative transcriptome analysis of the colon by RNA-seq show that the increased susceptibility phenotype of Irf1−/− mutants is linked to increased inflammation and immune cell infiltration in the colon of Irf1−/− mice, and is transferable by hematopoietic cells in bone marrow chimera experiments. Genes dysregulated in Irf1-deficient mouse colon are enriched in gene set hallmarks of active UC and IBD. In addition, analysis of The Cancer Genome Atlas (TCGA) database for IRF1 expression in human colorectal cancer patients revealed a statistically significant reduction of IRF1 in colorectal cancer patient tumors of Stage 1, 3, and 4. Together, these results suggest a critical role for IRF1 in regulating the type and intensity of inflammatory response in the colon, contributing to establishment of permissive conditions for progression of IBD to CA-CRC.We have tested a possible role of IRF1 in CA-CRC, using a combination of studies in mice and humans. We found that All animal experimentation was performed in accordance with the guidelines and regulations of the Canadian Council on Animal Care (CCAC) and were approved by the McGill University Animal Care and Ethics Committee.19, 8–10 week-old C57BL/6J, A/J, or Irf1−/− mice (The Jackson Laboratory) were injected ip with azoxymethane  followed by three 4-day cycles of 2% (w/v) DSS in the drinking water, with each cycle 17 days apart and euthanized 6 weeks later. In some experiments, mice were injected with AOM (7 mg/kg) once, followed by two 4-days cycles of 2% (w/v) DSS and sacrificed 3 weeks later. The model of AOM-induced colorectal cancer was as previously described20. For the chronic colitis model, mice were treated with three 4-days cycles of 2% (w/v) DSS and sacrificed after week 8.For the CA-CRC model21.Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin/eosin, were scored for markers of inflammation as previously described2 pieces, washed in 1X Hank’s Balanced Salt Solution (HBSS) containing 2% heat-inactivated fetal bovine serum (FBS), disrupted by vortexing, further treated with collagenase IV (20 mg/ml) and DNase I (10 mg/mL) in RPMI medium and passed through a 70 μM cell strainer to collect lamina propria cells. Cells were treated with blocking antibodies to CD16/32 (1:100), and stained for viability using Zombie Viability Dye V500 (1:400). Surface staining was done on ice for 20 min using anti-CD45 APC-eFluor780 , anti-CD11b APC , anti-Gr1 PerCp-Cy5.5 , anti-Siglec F PE , anti-CD3 FITC , anti-CD49b FITC , anti-B220 FITC , anti-CD4 PerCp-Cy5.5 , and anti-CD8a AlexaFluor 700 ,. Intestinal epithelial cells were collected as described in22. Cells were stained with anti-EpCAM PE-Cy7  and anti-FITC CD44 . Cells were analyzed on the Fortessa (BD Biosciences) and data analysis was performed on FlowJo (Tree Star).Colons were cut into ~1 cm23 and analyzed using publicly available bioinformatics work tools and software packages27. Differential gene analysis was evaluated as described28 where genes with <5 counts per million for at least 3 samples were filtered out. Genes were considered significantly differentially expressed if fold change was ≥1.5 or ≤−1.5 and FDR <0.01. Gene ontology (GO) enrichment analysis was performed using DAVID bioinformatics resources v6.829 and gene set enrichment analysis (GSEA) was analyzed using the GSEA software30. Gene sets from published data can be accessed at GSE4183. Relative abundance of certain transcripts was verified by quantitative PCR (qPCR), as described23. Primers for genes tested are listed in Supplementary Table 31 and were normalized to expression of Hprt.Mice received one injection of AOM D1) followed by one 4-day cycle of DSS  and were sacrificed at D26. Distal colons were collected and flash-frozen in liquid nitrogen. RNA was isolated from homogenized tissue with TRIzol reagent (Life Technologies) and purified using an RNeasy Mini Kit (Qiagen). RNA integrity was ascertained using a Bioanalyzer Total RNA Pico assay (Agilent). rRNA-depleted libraries were prepared using Kapa-stranded RNA-seq kit (Kapa Biosystems), sequenced as previously described followedIrf1−/− mice, fixed in 4% paraformaldehyde for 6 h at 4 °C, cryo-protected in 20% sucrose, embedded and frozen in optimal cutting temperature compound. Primary antibodies used were anti-CD11b , and anti-FITC Ki67 . Goat anti-rat secondary antibodies conjugated with DyLight 488 were used at a 1:1,000 dilution. Immunostained sections were counterstained with DAPI  to visualize nuclei. For quantification of proliferation, at least 5 fields were selected randomly, and all crypts were counted under 10X magnification. Crypts were considered “hyperproliferative” if Ki67+ cells extended from the base to at least 3/4 of the height of the crypts; the percentage of hyperproliferative crypts/normal crypts was calculated from total counts. All images were acquired on a Zeiss Axiovert 200 M Automated Inverted microscope.Colons were collected from untreated and D26 post-AOM/DSS-treated B6 and Irf1−/− mice, 8 weeks-old, were irradiated with 450 rad twice within a 3 hour interval on an X-Ray RS-2000 Biological Irradiator. After 3 hours, mice were injected intravenously with 5 × 106 red blood cell-depleted bone marrow from female donors. Mice received 50 mg/mL enrofloxacin (Baytril) in their drinking water 3 days prior to irradiation and for 3 weeks post-irradiation. Bone marrow reconstitution was verified at 8 weeks post-transplantation by flow cytometry. At 9 weeks post-engraftment, mice were subjected to AOM-DSS treatment as described above.CD45.1 and 32. Data are GC-RMA pre-processed log2 fluorescence intensities33 from Affymetrix Human Genome U133 Plus 2.0 Arrays. Intensities were quantile-normalized to reduce the inter-sample variations. Probesets with no or very low signals were filtered out, thus only probesets with a log2 fluorescence intensity of >5 for at least 3 samples were retained for subsequent analyses. Differential gene expression analysis was performed by comparing UC_act or UC_rem groups with the control group. Two statistical tests were applied to determine significance of differential expression: LIMMA and Bonferroni-corrected Student’s T-test; genes with a fold change ≥2 or ≤0.5 and with an adjusted p value ≤ 0.05 in both tests were considered statistically dysregulated.Raw microarray data generated from colon biopsies of patients with either active UC (UC_act) or those in remission (UC_rem), together with control non-inflamed tissue of UC patients and tissue from non-UC patients were obtained from Gene Expression Omnibus repository (GSE38713)34.Data were analyzed from the January 2016 version of the TCGA colon and rectal adenocarcinoma (COADREAD) dataset. When colon and rectal carcinomas were sequenced on both Illumina HiSeq & GA platforms, only the HiSeq results were used. Zero reads were replaced with a random number based on the mean and standard deviation of very low read numbers (with the number of reads <1). Two normalization methods were used, quantile normalization of all samples, and a Z-transformation of batches with at least 10 samples. For the Z-transformation, the overall mean of gene expression in each batch was Z-transformed to have a mean 0 and a standard deviation of 1. This gauged if a gene was expressed at a high or low level within this specific batch, and corrected for batch-specific effects. The batch-specific Z-filtered and quantile scores were used in all further survival calculationsIrf1−/− and B6 mice with human UC patients. Unpaired, two-tailed Student’s t-tests were used for all other determinations of significant differences between two groups.Except when otherwise indicated, data were analyzed with GraphPad Prism 5 software (GraphPad Software), and are presented as mean ± SD. The significance of weight loss measurements was evaluated by two-way ANOVA, followed by Bonferroni’s multiple-comparison test. Mann-Whitney test was used for analysis of histopathological scoring. A one-way ANOVA followed by Bonferroni’s multiple-comparison test was employed to compare proportions of intestinal proliferation. Fisher’s exact test was used to compare gene expression profiles of AOM/DSS-treated GEO: RNA-seq data: GSE116374.7 and colorectal cancer35, we tested the possible role of Irf1 in CA-CRC. For this, we compared the response of resistant C57BL/6J (B6) control and Irf1−/− mutant mice (on a B6 background) to AOM/DSS treatment  treatments with AOM, in absence of DSS treatment20  on colons from B6 controls and Irf1−/− mutants. Differentially expressed genes in Irf1−/− relative to B6 colons  was conducted  in common in both strains as well as genes dysregulated only in B6 or only in Irf1−/− mice  and the TNF-activating receptor family . GO terms for genes exclusively upregulated in Irf1−/− mutants included Inflammation and Leukocyte Activation, while B6 upregulated genes included Antigen Processing and Presentation and Innate Immune Response  where tumors are not yet present. Therefore, after a single treatment of AOM and DSS, the Irf1−/− mouse colons’ transcriptional profile is already enriched for genes upregulated in human IBD and human colorectal cancer.Comparative analysis of the response of B6 and ice Fig. . Analysinse Fig. . These aols Fig. . We obseIBD Fig. . GSEA anDSS Fig. . ImportaIrf1−/− mice might be associated with early alterations in epithelial proliferation. Immunofluorescence analysis of cell proliferation marker Ki67 revealed an increase of “hyperproliferative epithelial crypts” in the colons of both B6 and Irf1−/− mutants at D26, but in significantly greater abundance in Irf1−/− mutants  signature”38 in Irf1−/− mice post-treatment  preceding tumor emergence.We then investigated if the enriched human CRC signature in nts Fig. . FurtherD26 Fig. . These rent Fig. . ISCs arD26 Fig. . GloballIrf1−/− colons prior to and 26 days following treatment with AOM/DSS. While B6 controls showed a modest influx of CD45+ cells at D26, this influx was nearly three-fold greater in Irf1−/− mutants , primarily Gr1+CD11b+ neutrophils and inflammatory monocytes  chimeras and tested their susceptibility to AOM/DSS-induced CA-CRC susceptibility  confers significant CA-CRC susceptibility, at a level similar to that seen in Irf1−/− mutant animals receiving Irf1−/− mutant BM , possibly creating a tumor-promoting environment in these mice.Finally, to determine the overall contribution of this hematopoietic cells infiltrate to the CA-CRC susceptibility of ity Fig. . These s BM Fig. , whereasCRC Fig. . These rIRF1 in development of colonic inflammation and CA-CRC in humans, we first compared the AOM/DSS-induced transcriptional responses detected in the B6 and Irf1−/− mutant colons to that of published colon transcript profiles associated with human UC32. The extent of overlap  between genes upregulated in active UC and genes upregulated in the B6 and Irf1−/− datasets indicated that the Irf1−/− dataset had a greater and more significant overlap (28%) than the B6 datasets (22% overlap)  Fig. . In comp−/− Fig. , suggestIRF1 expression in human colorectal cancer patients revealed a statistically significant reduction of IRF1 in tumors of Stages 1, 3, and 4 of colorectal cancer patients  compared to controls (more prominent antigen presentation). Likewise, FACS immunoprofiling of AOM/DSS-treated B6 and Irf1−/− colons revealed a striking infiltration of myeloid cells  in Irf1−/− mice compared to B6 mice. Importantly, studies in bone marrow chimeras established that hematopoietic cells are the main driver of CA-CRC development in Irf1−/− mutants, as Irf1−/− BM can transfer CA-CRC susceptibility to control B6, while B6 BM can protect Irf1−/− mutants against CA-CRC.Studies by RNA-seq identified differential transcriptional changes in B6 vs. Irf1−/− mutants, which may correspond to early signs of persistent proliferation of intestinal epithelial cells.Other prominent transcriptional responses were associated with the differentiation and maturation of epithelial cells that may reflect a more curative response in B6. GSEA analysis also identified a stronger intestinal stem cell signature in Irf1−/− mutant. In this model, dysregulation of Irf1 causes a defect in myeloid and lymphoid cell lineages. Such deficiencies in the number and function of DCs, CD8+ T cells, and NK cells have been described for Irf1−/− mice43. Furthermore, our RNA-seq studies show that loss of Irf1 causes basal alterations in colonic immune and epithelial transcriptional programs  and phagocytotic markers  are increased, indicative of active pathogen detection. Furthermore, infiltration of pro-inflammatory cells in Irf1−/− colons is readily detectable by FACS analysis  based on cistrome analysis of pro-inflammatory transcription factors and IFN-γ–induced transcriptomes in myeloid cells, to prioritize positional candidates at complex inflammatory disease loci51. Analysis of the 20 genes of the IBD5 locus shows that IRF1 has the highest score (MIS ~ 15). These, together with our observation that Irf1 deficiency in untreated mice causes widespread transcriptional alterations of colonic immune and epithelial pathways provides compelling evidence that IRF1 is the “morbid” gene at the IBD susceptibility locus IBD5.In humans, we propose that reduced expression or partial deficiency in IRF1 may predispose to IBD and subsequent progression to colorectal cancer in those individuals based on several lines of evidence. Firstly, human Irf1−/− mutants shows more extensive overlap with genes dysregulated in active UC lesions than that of B6 controls, suggesting that, following tissue injury, Irf1 deficiency causes a pathological state in mice that resembles chronic inflammation seen in UC patients (Fig. Irf1−/− mutants identifies a highly statistically significant intersection with a human colorectal cancer transcript signature which is absent in B6 controls (Fig. Irf1 deficiency in the colon causes a pathological state in mice that resembles that seen in colorectal cancer patients. Finally, we note reduced IRF1 mRNA expression in tumors from stage 3 and 4 colorectal cancer (Fig. Secondly, examination of the transcriptional response of nts Fig. . Thirdlyols Fig. , and thicer Fig.  that is cer Fig. . Taken tSupplementary Information


Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, are a public health problem, causing millions of infections and killing almost half a million people annually. The ability of these pathogens to colonise almost every organ in the human body and cause life-threating infections relies on their capacity to adapt and thrive in diverse hostile host-niche environments. Stress-induced genome instability is a key adaptive strategy used by human fungal pathogens as it increases genetic diversity, thereby allowing selection of genotype(s) better adapted to a new environment. Heterochromatin represses gene expression and deleterious recombination and could play a key role in modulating genome stability in response to environmental changes. However, very little is known about heterochromatin structure and function in human fungal pathogens. In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of heterochromatin in regulating genome plasticity in the most common human fungal pathogens: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans.Human fungal pathogens, such as  Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans are the most common human fungal pathogens responsible for life-threating infections, especially in immune-compromised patients [Human fungal pathogens are a leading cause of human mortality worldwide and kill almost 2 million people every year . There apatients .During colonisation and infection of the host, human fungal pathogens encounter many different host environments to which they must adapt rapidly. For example, they must adapt to grow at high temperature  and they must survive the dramatic environmental changes following engulfment by macrophages and neutrophils of the host immune system. Indeed, following phagocytosis, fungal pathogens must resist reduced pH and the generation of toxic compounds such as hydrogen peroxide in the microcidal phagolysosomes .Similarly, during development of drug resistance, human fungal pathogens must survive the fungistatic and fungicidal effect of anti-fungal drugs. How do fungal pathogens respond rapidly to the continuous changes in the environment they encounter in the host and following drug treatment? Addressing this question is key to understanding how human fungal pathogens establish life-threating infections and develop resistance to anti-fungal drugs.In recent years, the importance of stress-induced genome instability has emerged as a key adaptive mechanism of human fungal pathogens . AlthougIt is unknown whether and how stress-induced genome instability in human fungal pathogens is regulated but it is likely that chromatin-mediated epigenetic regulation plays a key regulatory role in this process. Indeed chromosome rearrangements do not occur randomly and are more likely to occur at specific chromosomal locations enriched in DNA repeats ,13,16,17Saccharomyces cerevisiae and Schizosaccharomyces pombe, we still know little about the structure and function of repeat-associated chromatin in human fungal pathogens. Despite our detailed knowledge of heterochromatin structure and function in fungal model systems, such as In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of chromatin in regulating genome plasticity in human fungal pathogens.The eukaryotic genome is packaged into chromatin, which is composed of basic units called nucleosomes. Each nucleosome comprises two molecules each of histone H2A, H2B, H3 and H4, which form a spool to wrap 147 bp of DNA .Histones are globular proteins composed of a histone core, which mediates interactions between different histone proteins, and unstructured amino terminal tails, which extend out from the core nucleosome particle. Histone tails are subject to a wide variety of post-translational modifications, known as histone marks, that decorate distinct genomic regions and reflect different functional states . The enzChromatin can exist in two states: euchromatin and heterochromatin. Euchromatin assembles over gene-rich, non-repetitive DNA and it is associated with “active” histone marks permissive to transcription such as histone acetylation and methylation of lysine 4 on histone H3 (H3K4me) . Two different types of heterochromatin are assembled into distinct genomic regions. Constitutive heterochromatin (here simply termed heterochromatin) assembles at loci with low gene density and/or enriched in repetitive DNA where it silences the expression of most genes within or near it and promotes genome stability by suppressing unequal recombination events . FacultaIn mammalian cells, the most prominent chromatin feature of heterochromatin is global hypoacetylation, which leads to chromatin fibre compaction. In addition, specific “repressive” marks flag heterochromatic regions. A typical mark of heterochromatin is the methylation of histone H3 on lysine 9 (H3K9me) established by the H3K9 methyltransferases Su(var)3-9 . HeterocS. cerevisiae and S. pombe  arose from complete duplication of eight ancestral chromosomes followed by massive gene loss (nearly 90% of duplicated genes) and gene specialization  lacks most repressive histone marks, which are normally associated with mammalian heterochromatin. For example, the S. cerevisiae genome does not encode for Su(var)3-9 or Ezh2 orthologues and therefore its chromatin lacks the H3K9me and H3K27me repressive marks [S. cerevisiae has also no endogenous DNA methylation machinery and it is therefore devoid of 5mC DNA methylation [S. cerevisiae heterochromatin is the lack of active histone marks—most importantly histone H3 lysine 16 acetylation , a hallmark of DNA double-stranded breaks, is enriched at heterochromatin regions [ regions ,33. In c regions .S. cerevisiae heterochromatin assembles over genomic locations enriched in repetitive DNA: the silent mating-type (MAT) locus, subtelomeric regions and ribosomal DNA tandem arrays [As seen in other organisms, m arrays . The rolm arrays ,39,40,41MAT locus where, by controlling gene expression and recombination, it allows faithful mating-type switching, enabling sister cells to mate to produce a diploid. The MAT locus directs mating-type switching: a biological switch that allows haploid cells of one mating type to produce haploid cells of the other mating type [MAT locus contains either the a1 gene (MATa) or the α1 and α2 genes (MATα). These genes encode transcription factors that determine the expression of cell-type-specific genes necessary for pheromone production and detection. MAT shares homology with two other sites located on the same chromosome: HMLα and HMRa. Both loci have complete copies of the mating-type genes, but are kept transcriptionally silent by Sir2-dependent heterochromatin. Accordingly, haploid cells defective in heterochromatin express both sets of mating type genes at once and therefore are mating defective [Heterochromatin is assembled at the ing type . The MATefective ,40. MAT locus. This double-strand break is repaired by homologous recombination using the HMLα and HMRa loci as a donor, resulting in a unidirectional gene conversion event where the α or a gene from the HML/HMR locus is copied into the MAT locus. The recognition sequence for the HO endonuclease is present not only at the MAT locus but also at the HML and HMR loci. However, heterochromatin assembled at HML and HMR blocks access of the HO endonuclease to these loci, so cutting occurs only at MAT [MAT locus, heterochromatin promotes site-specific homologous recombination in addition to repressing gene expression at the HML and HMR loci.In wild-type (WT) cells mating-type switching is triggered by the HO site-specific endonuclease that specifically cleaves the y at MAT ,43. TherS. cerevisiae, subtelomeric regions contain several copies of X and Y’ non-coding DNA elements and are enriched for fast-evolving multi-gene families [S. cerevisiae [Subtelomeres are gene-poor and repeat-rich regions found adjacent to telomeric repeats at both ends of each linear chromosome . In S. cfamilies ,45. Subtrevisiae ,46.S. cerevisiae remains a mystery as its role in transcriptional repression and inhibition of genetic recombination seem to be minor. Indeed, even though subtelomeric heterochromatin can repress gene expression of marker genes integrated in proximity of certain X elements, only 6% of endogenous subtelomeric genes are silenced in a Sir-dependent manner [S. cerevisiae subtelomeric heterochromatin does not seem to play a major role in repression of mitotic recombination. Instead, genome stability at subtelomeres is promoted by the Ku70-Ku80 heterodimer, a protein complex involved in non-homologous end-joining and telomere end maintenance [Subtelomeric repeats are assembled into Sir2-dependent heterochromatin. The role of subtelomeric heterochromatin in t manner ,38. In antenance .The ribosomal DNA (rDNA) locus contains genes encoding for ribosomal RNAs (rRNAs) consisting of a tandem array of a ~9.1 kb unit repeated 100 to 200 times on chromosome XII. Each unit contains the 35S rRNA gene that is transcribed by RNA polymerase I and the 5S rRNA gene that is transcribed by RNA polymerase III. The 35S and 5S genes are separated by two non-transcribed spacers (NTS), also known as intergenic spacers (IGS), that contain three conserved elements: (i) an origin of replication (rARS), (ii) a replication fork blocking site (RFB) whose function is to avoid collision between replication and transcription machineries and (iii) a non-coding RNA promoter (E-pro) whose activity can be silenced by Sir2 ,47,48. TrDNA contraction is promoted by Sir2-dependent heterochromatin via repression of transcription from the E-pro promoter . In the Schizosaccharomyces pombe is a representative of the Taphrinomycotina subphylum of Ascomycota  machinery, chromatin writers and readers establishes and maintains this chromatin state  point centromeres, S. pombe contains large (35–110 kb) regional centromeres [otr, consisting of dg and dh repeats) coated in heterochromatin [Whereas tromeres . Fissionhromatin ,59,60. Phromatin . As a cohromatin . Pericenhromatin , and rephromatin .S. pombe MAT with its two silent cassettes, subtelomeric regions and the rDNA locus are assembled into transcriptionally silent heterochromatin that promotes genome stability. At the MAT locus, heterochromatin promotes mating-type switching by using a mechanism similar to that operating in S. cerevisiae [In addition to pericentromeric regions, the revisiae , subtelorevisiae  while rDrevisiae .S. pombe genome. These heterochromatin islands reversibly regulate gene expression in response to environmental changes. For example, during vegetative growth heterochromatic islands assemble at meiotic genes and are important for their transcriptional silencing [In addition to the large block of heterochromatin found at centromeres, the mating-type region, rDNA, and telomeres, smaller “heterochromatic islands” can be found across the ilencing , and othilencing .C. albicans belongs to the “CTG-clade” . However, clinical isolates exhibit karyotypic diversity, including aneuploidy and gross chromosomal rearrangements that can confer anti-fungal drug resistance due to altered copies of specific genes [C. albicans genome instability is induced by environmental conditions: the genome is relatively stable under optimal laboratory growth conditions but becomes more unstable under stress conditions. For example, chromosome rearrangements and/or aneuploidy dramatically increased in the presence of the anti-fungal drug fluconazole (100 fold) or at high temperature (10 fold) [in vivo mouse models [C. albicans is mainly an asexual organism lacking meiosis, genomic diversity is produced by mitotic genome instability [In the last decade, it became clear that genome plasticity is central to ic genes ,12,13. E10 fold) . Genome mes 2n = . Howevere models ,73. Givetability .Different types of DNA repeats are the major source of genome instability. These DNA elements include: (i) long, complex repetitive elements , (ii) loTLO genes [S. cerevisiae rDNA locus and consists of a tandem array of a ~12 kb unit repeated 50 to 200 times [Candida-specific DNA repeats composed of long tracts (10–100 kb) of nested DNA repeats that are found on 7 of the 8 C. albicans chromosomes [C. albicans MAT locus does not contain the S. cerevisiae-like HML and HMR cassettes for mating-type switching. In contrast C. albicans, possesses a single mating-type (MTL) locus on chromosome 5, which is normally heterozygous (a/α) in this diploid organism [Among the different types of repetitive DNA elements, long, complex repetitive elements are potentially perfect substrates for heterochromatin assembly. Long, complex repetitive elements are found at telomeric regions, the rDNA locus, and the major repeat sequence (MRS) . TelomerLO genes ,77. The 00 times . MRS locomosomes . The C. organism . S. cerevisiae, the C. albicans genome encodes for the histone deacetylase Sir2 but it lacks the H3K9 and H3K27 methylation machinery [C. albicans, although DNA methyltransferase orthologues cannot be identified by bioinformatic analyses [C. albicans genome encodes for components of the RNAi machinery  [C. albicans genome instability. DNA that is methylated on cytosine (5mC) has been detected at the rDNA locus and subtelomeric regions, suggesting that DNA methylation is an important contributor to C. albicans heterochromatin structure [C. albicans.Sir2-dependent heterochromatin assembles at subtelomeric regions and the rDNA locus where it dynamically represses expression of subtelomeric genes, leading to transcriptional noise and repression of non-coding RNAs originating for the non-trascribed spacer (NTS) regions of the rDNA locus ,83,84. Wnt (TRE) . The DNAtructure . It willin vivo mouse model system has demonstrated that in the host MRS are unstable genomic loci as they can expand and contract and are known sites of translocations [Given their highly repetitive nature, MRS repeats are expected to be ideal substrates for heterochromatin assembly. Surprisingly, Sir2-dependent hypoacetylated heterochromatin does not mark the MRS repeats. MRSs are assembled into highly acetylated chromatin that is permissive to transcription ,83. Howeocations ,86.C. albicans repetitive elements is remodelled upon entry into hostile environments such as those encountered during colonisation and infection of the host.It will be important to determine whether and how the chromatin state associated with Aspergillus genus belongs to the Pezizomycotina subphylum  to reach the lung alveoli. Inhalation of infectious conidia by immunocompromised individuals may lead to germination and growth of the fungus in the lung, followed by progression to serious diseases such as invasive pulmonary aspergillosis. Following inhalation, A. fumigatus adheres and penetrates the human respiratory epithelia and overcomes the response of surrounding cells particularly phagocytic cells, and therefore it must survive in a variety of host-niches environments.A. fumigatus has a haploid genome and possesses a fully functional sexual reproductive cycle [MAT locus that, similarly to C. albicans and C. neoformans, does not possess a S. cerevisiae-like silent-cassette system.ve cycle . Mating A. fumigatus isolates and harbour many gene clusters required for synthesis of secondary metabolites (SM): small molecules that are not essential for life but are important virulence factors [A. fumigatus strains revealed an incredible intra-species genome variation of SM gene clusters with SNPs, gene deletions, gene amplifications and reciprocal translocations [Genetic diversity associated with subtelomeric regions might be important for driving adaptation to different host niche environments. Subtelomeric regions are the most diverse genomic regions across different  factors ,92,93. Iocations . A. fumigatus is still largely unexplored, studies performed in A. nidulans and other Aspergillus species strongly suggest that heterochromatin is a critical regulator of subtelomeric SM gene clusters.Although heterochromatin structure and function in Aspergillus is marked by hypoacetylated nucleosomes that are methylated on H3K9, while no H3K27 methylation system has been identified [A. flavus [Aspergillus species contain an active RNAi machinery that has an anti-viral defence role but it is unknown whether RNAi contributes to heterochromatin formation 3-9 and of the histone deacetylase Hda1 leads to increased levels of SM in cycling cells [A. fumigatus contains an active H3K9 methyltransferase ClrD/Su(var)3-9 and an orthologue of Swi6/HP1 (Afu6g07550). Deletion of the A. fumigatus CLRD gene results in developmental defects but its contribution to subtelomeric secondary metabolite gene clusters expression has not yet been investigated [A. fumigatus heterochromatin structure contributes to SM gene expression regulation and the genome plasticity of SM gene clusters. In ng cells ,102. A. stigated . It willCryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths annually worldwide 3-9 and of the H3K27 methyltransferase Ezh2. In addition, C. neoformans possesses 5mC DNA methylation that is dependent on the sole cytosine DNA methyltransferase Dnmt5 [C. neoformans lacks silent mating-type cassettes and does not switch mating type [The hromatin . Indeed,se Dnmt5 ,115 and se Dnmt5 . Combinaing type .C. neoformans contains large regional centromeres (20–40 kb) that are gene-poor and enriched in retrotransposons [C. neoformans as, unlike what is observed in S. pombe, RNAi null mutant strains are not sensitive to the microtubule destabilising drug TBZ, indicating that faithful chromosome segregation does not require the RNAi machinery [C. neoformans RNAi-mediated gene silencing, such as sex-induced silencing, is post-transcriptional and is not associated with H3K9 methylation [nsposons ,119,120.nsposons . The rolachinery . Finallyhylation .A different combination of heterochromatic marks is detected at subtelomeric regions. Low levels of H3K9me can be detected at subtelomeres and H3K9me is probably responsible for targeting 5mC DNA methylation to these regions. In contrast, high levels of H3K27 methylation, a chromatin mark that is in general associated with facultative heterochromatin, is detected at subtelomeres ,115,122.C. neoformans centromeric and telomeric regions and it will be important to investigate whether and how these chromatin states contribute to genome instability in canonical and titan cells.Thus, different types of heterochromatin assemble at S. cerevisiae and S. pombe, which can be easily adapted for human fungal pathogens.Data from many different laboratories have highlighted how heterochromatin structure is different in different fungal organisms. Given that heterochromatin is likely to play key regulatory roles in stress adaptation, there is a real need to investigate the chromatin state associated with DNA repeats in human fungal pathogens. Investigation of heterochromatin structure and function in this group of medically relevant microorganisms is facilitated by the robust methods developed in the fungal model systems S. cerevisiae and S. pombe. To distinguish between nucleosome occupancy and depletion/enrichment of specific histone modifications, ChIP-seq should also be conducted with antibodies that recognize unmodified histones. Similarly, 5mC DNA methylation can be detected at single base-pair resolution by bisulfite genomic sequencing [Genome-wide maps of active and repressive histone marks have been instrumental in identifying heterochromatin regions across eukaryotic genomes ,123. Sucquencing .ChIP-seq has one major drawback: it requires a priori knowledge of the type and position of the histone modification of interest. It is possible that repeat-rich genomic regions of human fungal pathogens are assembled into a chromatin state marked by novel histone modification(s). Novel histone modifications could be identified by performing mass spectrometry (MS)-based proteomics . ChIP-se+URA3) is integrated at heterochromatic regions [+URA3 marker gene renders cells resistant to 5-fluoroorotic acid (5-FOA). Similarly, genomic instability at repeat-rich regions can be assessed genetically by for example measuring loss of heterozygosity (LOH) rates of a heterozygous marker gene inserted at repeat-rich regions or by measuring gene conversion between heteroalleles of an easily scored nutritional marker [Heterochromatin function can also be investigated by adapting simple and affordable genetic systems to human fungal pathogens. The transcriptionally repressive state of heterochromatin regions can be assessed by developing reporter gene silencing assays where a reporter gene (such as  regions  Transcril marker ,127. TheChromatin structure and function in human fungal pathogens is still a poorly investigated field of research. However, the limited analyses that have been performed have highlighted how heterochromatin is often atypical in human fungal pathogens as the basic building blocks of heterochromatin are rearranged in this diverse group of medically important microorganisms. Given that similar structure often equates to different functions, it is dangerous to directly extrapolate findings from yeast model systems to human fungal pathogens. Heterochromatin and its ability to rapidly and reversibly regulate gene expression and genome instability could be a key regulatory mechanism underlying virulence and pathogenicity of human fungal pathogens. It is therefore surprising that we still know very little about chromatin structure and function in this medically important microorganisms and future research will be instrumental in unveiling its role in virulence and pathogenesis.


SLC20A2 is encoding the phosphate transporter PiT-2 and was identified in 2012 as the causative gene of familial IBGC. In this study, we investigated functionally two novel SLC20A2 variants  and two SLC20A2 variants  previously reported from patients with IBGC. We evaluated the function of variant PiT-2 using stable cell lines. While inorganic phosphate (Pi) transport activity was abolished in the cells with c.82G > A, c.358G > C, and c.1487G > A variants, activity was maintained at 27.8% of the reference level in cells with the c.680C > T variant. Surprisingly, the c.680C > T variant had been discovered by chance in healthy members of an IBGC family, suggesting that partial preservation of Pi transport activity may avoid the onset of IBGC. In addition, we confirmed that PiT-2 variants could be translocated into the cell membrane to the same extent as PiT-2 wild type. In conclusion, we investigated the PiT-2 dysfunction of four SLC20A2 variants and suggested that a partial reduced Pi transport function of PiT-2 might not be sufficient to induce brain calcification of IBGC.Idiopathic basal ganglia calcification (IBGC) is a rare intractable disease characterized by abnormal mineral deposits, including mostly calcium in the basal ganglia, thalamus, and cerebellum.  IBGC is characterized by abnormal mineral deposits, mostly calcium, in brain regions, including the basal ganglia, thalamus, cerebellum, and others. We have investigated the patients in Japan5. We have 231 patients with 33 familial case now in Japan. SLC20A2 (IBGC1 was previously named IBGC3) encoding the phosphate transporter PiT-2 was first identified in 2012 as the first causative gene of IBGC6. Thereafter, other causative genes of IBGC were continuously discovered, such as PDGFRB (IBGC4), PDGFB (IBGC5), and XPR1 (IBGC6)9. Most recently, MYORG is identified as a new causative gene of IBGC and is associated with recessive IBGC10. Among these, IBGC1 is the most frequent and accounts for about 30%–50% of familial cases5. Currently, no effective therapy is available for patients with IBGC2.Idiopathic basal ganglia calcification (IBGC), also known as Fahr’s disease or prevailingly as primary familial brain calcification, is a rare neurodegenerative disease. Historically, too many nomenclatures have been discussed and used for this conditionSLC20A2 is a sodium-dependent phosphate type III transporter belonging to the inorganic phosphate transporter (PiT) family11. This membrane protein consists of 12 transmembrane domains and transports inorganic phosphate (Pi), especially in the brain14. Several groups, including our group, have reported novel variants in SLC20A220. Before the discovery of SLC20A2 variants in patients with IBGC, Bottger, P. and Pedersen, L. had shown that the artificial replacements E55K, E575K, D28N, and D506N were leading to PiT-2 variants unable to transport Pi into the plasma of Xenopus laevis oocytes22.PiT-2 encoded by SLC20A2 variants have suggested that Pi homeostasis is disrupted as a result of Pi transport dysfunction of PiT-2 causing IBGC23. Wang et al. showed that the six variants, S601W, S601L, T595M, E575K, G498R, and V42del, of SLC20A2 in patients with IBGC caused a Pi transport dysfunction of PiT-2 using Xenopus laevis oocytes6. Furthermore, Yamada et al. showed that the function of PiT-2 was impaired by the A51V, R71H, T115M, and S637R variants using PolyPhen-24. Recently, Sekine et al. demonstrated a remarkable decreased activity in 32Pi intracellular transport using induced pluripotent stem cells (iPSCs) from patients with the SLC20A2 variant encoding W616X23. Many SLC20A2 variants have been reported so far, but no hotspot of variant sites has been specifically identified24.Several studies on 11-L161 (N-PD1131) and V492-V640 (C-PD1131) located at the N-terminus and C-terminus of PiT-2, respectively, had been identified as potentially important for maintaining Pi transport activities25. Based on this finding, Bottger, P. and Pedersen, L. have shown the importance of PD1131 for maintaining Pi transport activities with artificial replacement or deletion in PiT-226.The ProDom regions I29.PiT-2 also functions as a receptor for amphotropic murine leukemia virus (A-MuLV), but no correlation between viral receptor activity and phosphate transporter activity has been reportedSLC20A2 and established stable PiT-2-expressing cell lines with the two novel variants and with two variants reported previously30 to analyze their function in PiT-2 and explore the pathophysiological mechanisms of IBGC. Furthermore, we discuss the possibility of the partial decrease in Pi uptake might not be sufficient for the onset of IBGC.In the study, we report two novel variants in The clinical manifestations are summarized in Table SLC20A2 variant  in exon 6 was accidentally found in his wife, son, and daughter, but not in the proband himself . Her DNA analysis has not been performed yet.The patient was an 81-year-old woman and had been referred to the department of neurology for evaluation because of colon cancer. She had suffered from right frontal subcortical hemorrhage 3 years before. She presented with left superficial sensory impairment after the stroke and postural hand tremors. Her CT images revealed severe calcification at the bilateral basal ganglia, mild calcification at the cerebellum dentate nucleus, and faint calcification at the pineal bodies, the choroid plexus, and subcortical white matter Fig. . Her DNASLC20A2 have been reported in our previous study30. Briefly, this 63-year-old woman presented with bilateral drop feet and gait disturbance due to peripheral neuropathy. The neurological examination showed mild cognitive impairment (MMSE 21), cerebellar ataxia, and spasticity in lower extremities. Her CT images revealed severe calcification at the bilateral basal ganglia and subcortical area, and faint calcification at the thalamus, but none at the cerebellum dentate nucleus . Her DNA study showed a variant  in exon 3. Her son had similar clinical manifestations and CT images  within 1 week. Her CT images revealed severe calcification at the bilateral basal ganglia and moderate calcification at the cerebellum dentate nucleus  gene was introduced into the viral vector to co-express GFP protein in Flp-In CHO cells infected with A-MuLV  and the region of V492-V640 (C-PD1131) were emphasized with orange and blue, respectively. Variants reported here are shown in red. Whereas PiT-2-D28N, G120R, and C496Y variants were found in the PD1131 region, the PiT-2-A227V variant was not. . The region of Iot. Fig. .Figure 5SLC20A2 . The four missense variants were predicted to have deleterious damages of protein function in the in silico analysis using PolyPhen-24. In addition, missense variants showed impairment in the Pi transport activity in CHO cells stably expressing them and in endothelial cells developed from patient’s iPSCs23.We have previously reported six new variants in SLC20A2  with their clinical manifestation. One new missense variant was found by chance in the clinically non-affected persons of the family 1 genetic study. Four variants  were predicted to cause deleteriousness of protein function based on the PolyPhen-2 result. We performed a functional study using Flp-In CHO cells expressing PiT-2 variants.In this study, we have reported two new missense variants in Interestingly, the Pi transport activity in PiT-2-A227V cells was maintained at 27.8% of the level of PiT-2-WT cells, whereas in D28N, G120R, and C496Y variants, the activities were lower than 10% of the control level. These results indicate that PiT-2 dysfunction caused by variants leads to the pathogenesis of IBGC and suggest that a partial decrease in Pi uptake might not be sufficient for the onset of IBGC.11. A single nucleotide variant of SLC20A2 from IBGC patients can cause loss of Pi transport activity15. The PD1131 region in the N-terminus and the C-terminus of PiT-2 was reported as essential for Pi transport activity26, and each single variant of SLC20A2 reported previously6 was found in the PD1131 region. Among the variants studied, PiT-2-D28N and G120R are located in the N-PD1131 region on the N-terminal side, and PiT-2-C496Y is located in the C-PD1131 region on the C-terminal side. Three variants of this study influenced the Pi transport activity and manifested typical clinical features resulting from phosphorus homeostasis impairment. On the other hand, the single nucleotide variant A227V, found in the IBGC patient’s families presenting no IBGC clinical features, showed 27.8% of the normal Pi transport activity.PiT-2 is a 12-pass transmembrane protein and sodium-dependent phosphate transporter of type III26. The differences between A227V variant and L183-R254 deletion26 are likely derived from the different mechanisms of their transporter activities.A previous report had shown that deletion of the L183-R254 region of PiT-2, encompassing the A227V variant position, showed a diminished the Pi transport activityInterestingly, the retention of the Pi transport activity in the A227V variant was correlated with no clinical manifestations and no findings in brain CT images, suggesting that a partial decrease in Pi uptake might not be sufficient for the onset of IBGC.SLC20A2 variants found in IBGC diminished the Pi transport activity, the patients carried heterozygous variants, and both wild-type PiT-2 and variant PiT-2 are theoretically expressed in the body. The mechanism by which SLC20A2 variants lead to IBGC has not yet been elucidated. SLC20A2 variants could lead to either haploinsufficiency or dominant negative effects. Based on the haploinsufficiency hypothesis, half of SLC20A2 expression would decrease the Pi transport activity32. On the other hand, a study suggested the dominant negative effect of the SLC20A2 variants encoding D28N, H502A, and E575K on the Pi transport activity33. The effect of SLC20A2 variants in IBGC remains, however, controversial. The higher levels of Pi detected in CSF of patients with IBGC, especially SLC20A2 variants, suggest the disease progresses with the dyshomeostasis of Pi transport in the brain.In this study, although the 29. However, the correlation between viral receptor activity and Pi transport remains unclear. We assessed the gamma-retroviral receptor function and found that although viral receptor functions were altered by variants of PiT-2, the activity varied between 50% and 160% of WT levels in all variants. Furthermore, the translocation of PiT-2 to the cell surface was assessed using the HiBiT technology to measure the membrane transfer rate of WT PiT-2 and PiT-2 variants. The membrane translocation rate of PiT-2-D28N was significantly increased, whereas those of G120R, A227V, and C496Y were similar to WT. Although the virus receptor function mediated by D28N seems related to membrane surface relocation, the relationships among virus receptor function, Pi transport activity, and/or clinical symptoms remain to be elucidated.The originally reported PiT-2 function was as a retrovirus receptor on the cell surface35. However, the relationship between these functions and IBGC pathology has not been recognized. The neurite outgrowth is thought to be associated with loop 7, which is a large intracellular domain of PiT-234. In addition, Kimura et al. have shown that in patients carrying the S637R variant of PiT-2, the expression level of PiT-2 is clearly low in the regions of high PiT-2 expression, including the frontal cortex, putamen, and cerebellum36. Taglia et al. have shown that in PiT-2 trp626_629dup patients, PiT-2 does not migrate to the membrane and is abnormally localized to the nucleus37. Yamada et al. have suggested that clinical features in patients with IBGC are similar according to the variant sites. In Case 3, both the proband and her son showed neuropathy. Whereas the diverse effects of PIT-2 variants should be clarified, other factors, such as environment and disease-modifying genes, should be considered.In addition, PiT-2 was reported to have additional functions, including sensing extracellular Pi concentrations and modulating neurite outgrowthIn conclusion, we have reported two novel pathogenic variants in PiT-2 and showed their associated dysfunction using CHO cells. Interestingly, in the A227V variant, the Pi transport activity partially remained, and no clinical manifestations of IBGC were found in the carriers. On the other hand, this suggests that the partial increase of PiT-2 activity may lead to improved manifestation in patients with IBGC.4. Briefly, patients with causative biochemical abnormalities, including calcium, inorganic phosphate, and intact parathyroid hormone (iPTH), were excluded. The genetic survey and all experiments on human blood samples were approved by the Ethics Committees of Gifu University and Gifu Pharmaceutical University and performed in accordance with Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan, and Ethical Guidelines for Human Genome/Gene Analysis Research in Japan. After written informed consent was obtained, peripheral blood samples were collected. This study was registered to the UMIN Clinical Trials Registry approved by International Committee of Medical Journal Editors (UMIN000030100).We collected clinical information on patients with IBGC in a nationwide study supported by a grant for research on intractable diseases from the Ministry of Health, Labor and Welfare of Japan. The diagnostic criteria were described previouslyGenomic DNA was extracted from the patient’s blood samples using DNA Quick II . PCR amplification was performed using the primers whose sequences are listed in Supplementary Table The CHO and Flp-In CHO cell lines were cultured in Ham’s F-12 medium supplemented with 10% (v/v) fetal bovine serum according to the manufacturer’s protocol (Thermo Fisher Scientific). The Platinum-A (Plat-A) cell line was cultured using Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) FBS.SLC20A2 cDNA (NM_006749) was purchased from Thermo Fisher Scientific and subcloned into a p3 × FLAG-CMV-14 vector (Sigma-Aldrich) with the flag tag inserted at C-terminal between the HindIII/EcoRI sites (PiT-2-flag vector). The SLC20A2 with the flag tag was subcloned into the pcDNA5/FRT vector between the HindIII/NotI sites . Subsequently, the SLC20A2 variant  was generated using the PrimeSTAR® Mutagenesis Basal Kit (Takara Bio Inc.) according to the manufacturer’s protocol. For HiBiT analysis, the SLC20A2 variant  and wild-type SLC20A2 were transferred into the pBiT3.1-C vector (Promega) between the HindIII/EcoRI sites using restriction enzymes. The primer set sequences are listed in Supplementary Table Human Flp-In CHO cells were seeded in six-well plates and co-transfected with pOG44 and pcDNA5/FRT-PiT-2-flag  or GFP (mock) according to the manufacturer’s protocol (Thermo Fisher Scientific). Hygromycin B-resistant cells were selected and cloned for experiments.Cells were washed with PBS and lysed in 200 µL RIPA buffer . Cell extracts were incubated on ice for 20 min and then centrifuged at 14,000 × g for 30 min at 4 °C. Supernatants were collected as protein samples, mixed with SDS sample buffer and incubated overnight at 4 °C. Protein concentration was determined using the Pierce BCA protein Assay Kit (Thermo Fisher Scientific). Proteins were separated by 10% SDS-PAGE for 50 min and transferred to PVDF membrane for 90 min. Blocking was performed with Blocking One  for 1 h at room temperature (RT). After blocking, the membrane was incubated at 4 °C overnight with the following mouse monoclonal primary antibodies: ANTI-FLAG M2  and β-actin  dissolved in 5% Blocking One/TBS-T , and 5% skim milk, respectively. After incubation, the membrane was incubated with the goat anti-mouse secondary antibody conjugated with HRP  dissolved in 3% Blocking One/TBS-T or 3% skim milk for 60 min at RT. The membrane was incubated in ECL Prime  to generate chemiluminescence from HRP antibodies, and chemiluminescence was detected using LAS3000 Mini (Fujifilm). Band density was measured using ImageJ software.38. The transport rate was expressed as nmol Pi per minute per mg protein.All phosphate uptake assays were performed essentially as describedPlat-A cells were seeded in 10 cm dishes and transfected with pMXs-EGFP. Cell culture supernatant was collected and added to the Flp-In CHO cells. Cells were lysed in PBS. The amphotropic receptor function was analyzed by measuring GFP expression cell count rates using a BD FACSVerse flow cytometer (BD Biosciences).via the co-transfection of GFP. Nano-Glo HiBiT Extracellular Reagent was prepared in advance and added to the transfected CHO cells. Cells were incubated for 10 min, and luminescence of the membrane protein was measured using the GloMax Multi Detection System (Promega). After measurement, cells were lysed with Triton-X100, and total protein was measured.CHO cells were seeded in 96-well plates and transiently transfected with pBiT3.1-C-PiT-2 . Translocation rates of PiT-2 were measured using the Nano-Glo HiBiT Extracellular Detection System according to the manufacturer’s protocol (Promega). Briefly, HiBiT is a small tag protein of 11 amino acids that binds to LgBiT, and the HiBiT–LgBiT complex emits luminescence upon substrate binding. Because LgBiT is impermeable to the cell membrane, only the HiBiT–LgBiT complex on the cell membrane surface is detected. The membrane translocation rate was calculated as the ratio of the membrane protein expression level to the total protein expression level. The transfection efficiency of each vector was confirmed post hoc comparisons was performed using the Bonferroni/Dunn test (Stat View).Data are given as the mean ± standard error of the mean (SEM). The significance of differences was determined by an analysis of variance (ANOVA). Further statistical analysis for Supplementary Information accompanies this paper at doi:Supplemental information


Previously, we developed a widely used method, BETA, to integrate TF ChIP-seq peaks with differential gene expression (DGE) data to infer direct target genes. Here, we provide Cistrome-GO, a website implementation of this method with enhanced features to conduct ontology analyses of gene regulation by TFs in human and mouse. Cistrome-GO has two working modes: solo mode for ChIP-seq peak analysis; and ensemble mode, which integrates ChIP-seq peaks with DGE data. Cistrome-GO is freely available at  Insight into the biological roles of transcription factors (TFs) can be acquired through analyses of the ontologies of the genes that they regulate. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), is a broadly used technique for identifying TF binding sites genome wide . DespiteTo avoid the limitation of binary value assignments, we have previously developed a model that integrates the number of ChIP-seq peaks and their distances from a gene’s TSS in a real valued estimate of the TF’s potential for regulating that gene . In addi0d is the decay distance of the weight function, with a default value of 1 kb for promoter-dominant TFs and 10 kb for the enhancer-dominant TFs. All k peaks near the TSS of gene g (within the distance of 150d) are used in the calculation, and id is the distance between the ith peak's center and the TSS. In the advanced settings users can override the default decay rate parameter 0d.The RP score of a gene reflects the likelihood of the TF being a direct regulator of that gene. In Cistrome-GO, the RP score of each gene is defined as the weighted sum of peak contributions, where the weights decay exponentially with distance to the TSS Figure . The parRPR is derived by decreasing adjusted RP score.Some genes tend to display higher RP scores than others across many TF cistromes. To quantify this bias, we calculated RP scores for each gene for 8470 and 6243 TF ChIP-seq datasets in human and mouse, respectively. For each ChIP-seq dataset, the RP scores for all genes were scaled to the range in  by min–max normalization. We define the baseline RP score of a gene as the median value of its scaled RP scores in all processed ChIP-seq data in human and mouse, respectively. The genes with higher baseline RP scores are enriched in certain biological processes, such as regulation of intracellular signal transduction . To mitihttp://go.cistrome.org/. Cistrome-GO was implemented using the Django framework and an HTML/JavaScript interface. The source code is available at https://bitbucket.org/liulab/cistrome-go/src/master/. Cistrome-GO has two working modes. If the user provides both a TF’s ChIP-seq peak file in Browser Extensible Data (BED) format (such as MACS2 (Cistrome-GO is freely available at as MACS2  output) as MACS2  output),as MACS2 . If the as MACS2 .10) within 1 kb of the TSS. If any of the calculated percentages is larger than a threshold, the TF will be classified as promoter-dominant. Otherwise, it will be classified as enhancer-dominant. We calculated the percentage of peaks close to TSS for 8470 human TF ChIP-seq data. Based on the distribution of the percentages, we set 20%  as the threshold between promoter- and enhancer-dominant TFs. In Cistrome-GO, promoter-dominant and enhancer-dominant TFs have different parameters to calculate RP scores by default. Users can override these default settings in the advanced options.A TF can regulate the expression of its target genes by binding cis-regulatory elements, including promoters and enhancers. For some TFs, such as E2F1, most binding sites are close to the TSS of target genes (<1 kb). Such TFs are of the promoter-dominant type. On the other hand, the binding sites for TFs such as AR and ESR1 tend to be less enriched in promoters, and those TFs are of the enhancer-dominant type. When a user uploads a TF’s ChIP-seq peak file, Cistrome-GO calculates the percentages of the most significant ChIP-seq peaks  are integrated by rank product. For example, if the ChIP-seq and DGE ranks of gene i are RiRP and RiDE, respectively, its rank product score is iRRP RiDE. The genes at the top of the list tend to have higher adjusted RP scores, together with more significant gene expression changes upon the TF’s perturbation; therefore, they are more likely to be true direct targets of the TF. In the settings users can opt to analyze upregulated genes, downregulated genes, or all genes. In the ensemble mode, Cistrome-GO also reports a receiver operating characteristic curve and precision-recall curve to assist the users to evaluate the association between ChIP-seq peaks and differential expressed genes. These curves are calculated on the basis of differentially expressed gene defined true positives and true negatives, with predictions based on adjusted RP scores.If the user provides a DGE analysis file in addition to a ChIP-seq peak file, an additional gene rank P-value and FDR is calculated for each GO or pathway term, and only the terms and pathways with FDRs <0.2 are reported.In the ensemble mode, the gene rank is based on the rank product score, while in the solo mode, the gene rank is based on the adjusted RP score. In both modes, the top ranked genes are most likely to be directly regulated by the TF. In order to determine the GO terms ,12 and Khttps://doi.org/10.6084/m9.figshare.8006285.To compare functional enrichment results between Cistrome-GO and published web servers in a quantitative way, we compiled a list of standard Biological Process (BP) GO terms for the evaluated TFs as follows. Among the BP terms annotating the evaluated TF in AmiGO, we removed the processes with more than 2000 genes and <10 genes, and the remaining ones are regarded as the standard BP terms for the TF. As the set of GO terms used in Cistrome-GO is not identical to those used in other web servers, to make a fair comparison of their performance, the comparisons presented in this manuscript are limited to BP terms within the GO set used in Cistrome-GO. Given a functional enrichment analysis from Cistrome-GO or any of the other websites, the 10 most significant BP terms in a GO set used in Cistrome-GO are selected. For each of these top enriched terms, the semantic similarity between it and each of the standard BP terms is calculated using GOGO , and theTo demonstrate the use of Cistrome-GO, we first display its output in solo mode (i.e. without integration of DGE data). MYOD1 is known to function as a master regulator in muscle cell differentiation, and most of its annotated BP GO terms are related to muscle development. Given a MYOD1 ChIP-seq peak file from myoblast , Cistromhttps://doi.org/10.6084/m9.figshare.8006198). For each TF ChIP-seq dataset, we used the top 5000, 10 000, 15 000, 20 000 and all peaks ) of the ChIP-seq peak file as input for each web server separately, and we calculated MSS scores between web server reported top 10 enriched BP terms and the standard BP terms . Based on the comprehensive comparison, the median performance of Cistrome-GO is better than that of ChIP-Enrich and Enrichr, and it is comparable to that of GREAT . For each TF, if there is more than one available ChIP-seq dataset, the one with the largest number of peaks was used in the comparison. We compiled a list of standard BP terms for each TF , and the TFs with over 10 standard BP terms were kept. In total, 256 TF ChIP-seq datasets were used in the comparison  to the TSSs of genes . For promoter-dominant TFs, the decay distance used to calculate adjusted RP scores is defined to be smaller than for the enhancer-dominant ones to recapitulate their promoter-related regulatory role. In Cistrome-GO, the default decay distance parameter is 1 kb for promoter-dominant TFs and 10 kb for enhancer-type TFs. In this study, an E2F1 ChIP-seq peak file from a prostate cancer cell-line LNCaP  was usedPreviously, we developed a software package BETA, which combined the RPs defined by ChIP-seq peaks with relevant DGE data to improve the inference of the TF’s direct target genes . In CistDespite the popularity of ChIP-seq technology and the importance of functional enrichment analysis, few tools are available for functional enrichment analysis based on ChIP-seq peaks. Cistrome-GO has three unique features compared to published web servers. First, Cistrome-GO is the only tool to use continuous values instead of binary values to measure each gene's potential to be regulated by a TF based on ChIP-seq peaks. Tools based on binary value assignments of peaks to genes could fail to identify the relevant GO terms in cases with many ChIP-seq peaks, as they tend to assign too many genes as TF targets. Second, Cistrome-GO is the only tool with an option to integrate differential expression data upon a TF’s perturbation to improve the inference of the TF’s direct target genes. Third, Cistrome-GO performs GO term enrichment analysis based on gene rank instead of an unordered list of genes. In this way the genes are ranked by their likelihood of being the direct TF targets, instead of being selected using an arbitrary cutoff (e.g. within 10 kb of the TSS).Cistrome-GO is a powerful and user-friendly website for functional enrichment annotation of TF regulation. With TF ChIP-seq data, and optional DGE data, Cistrome-GO outputs a table of gene RP ranks, and tables of GO and pathway enrichment. In summary, Cistrome-GO can give biologists a deeper understanding of TF function.gkz332_Supplemental_FileClick here for additional data file.


Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and  For microarray and chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, data were reanalyzed from previous studies, and from a new microarray dataset  and subventricular zone (SVZ) of E14.5 mouse neocortex contain four cell types: RGPs, apical IPs (aIPs), basal IPs (bIPs), and postmitotic projection neurons (PNs) , and expressed mainly in VZ; aIP genes were enriched in Tbr2-GFP+ cells , and expressed mainly in VZ; bIP genes were enriched in Tbr2-GFP+ cells, and expressed mainly in SVZ; PN genes were enriched in Tbr2-GFP+ cells, and expressed in IZ/CP. Some neuronal differentiation genes were expressed by not only neurons, but also progenitor cells undergoing neuronal differentiation. Also, some neuronal genes were widely expressed in forebrain neurons, while others were restricted to cortical PNs. Thus, neuron-specific genes were further classified according to initial zone of expression , and specificity for cortical or general neurons. If different microarray probes for the same gene showed enrichment in Tbr2-GFP+ and Tbr2-GFP− cells (“conflicted” probes), the gene was not considered specific for cell type. Genes with rostrocaudal expression gradients were identified, and classified according to zone of expression, as previously described , Tbr2 conditional knockout (cKO), and Tbr1/2 double KO/cKO (dKO) mouse embryos were produced as described . On E14.5, embryos were harvested, and neocortex was immediately dissected and frozen as described . Each embryonic neocortex was an independent biological replicate. The number of samples (n) of each genotype was: 3 control, 4 Tbr1 KO, 2 Tbr2 cKO, and 3 Tbr1/2 dKO. The microarray results were analyzed statistically as described .This study was carried out in accordance with the recommendations of Guide for the Care and Use of Laboratory Animals, National Research Council. The protocol was approved by the Institutional Animal Care and Use Committee of Seattle Children's Research Institute.Previous ChIP-seq raw data were obtained and reanalyzed for Pax6  in TF mutant neocortex compared to control on microarray. For analysis of Tbr1 and Tbr2 direct target genes, differential expression (p < 0.05) on either MA1 or MA2 was accepted as evidence of regulation. Genes regulated synergistically by Tbr1 and Tbr2 were identified by the presence of both Tbr1 and Tbr2 binding sites, and significant differential expression (p < 0.05) in Tbr1/2 dKO cortex, but not in Tbr1 KO or Tbr2 cKO cortex independently.Genes were defined as direct targets of Pax6, Tbr2, or Tbr1 regulation if the gene showed both TF binding by ChIP-seq, and differential expression  and PNs, respectively , while ISH showed Pax6 in both VZ and SVZ , we first examined whether Pax6, Tbr2, and Tbr1 transcriptionally regulate each other and/or themselves.Tbr2 expression  neocortex, expression of Pax6  was greatly increased , while Tbr2 was greatly decreased .Previous studies have found that Pax6 directly represses its own transcription . In contrast, Tbr1 was downregulated in Tbr2 cKO cortex. We also noted Tbr2 binding to its own gene (Tbr2), although the functional effects were uncertain: Tbr2 mRNA expression is reduced due to Tbr2 cKO . Thus, Tbr1 does not appear to directly regulate Tbr2, Pax6, or Tbr1.ChIP-seq analysis of Tbr1 showed that Tbr1 binds to the Together, these data indicate that the Pax6→ Tbr2→ Tbr1 cascade operates as a positive feedforward cascade, but also self-regulates by direct negative feedback effects Figure .Tbr1 locus that persist in postmitotic neurons. For example, removal of repressive histone marks by Jmjd3, an interacting protein of Tbr2, may create a permissive chromatin environment for Tbr1 transcription —except for overlapping expression of Pax6 and Tbr2 in some IPs , and can be reversed (erased) by pathways involving Gadd45a/b/g, Tet, and Aicda genes , and zinc-finger proteins such as Kaiso (Zbtb33), Zbtb4, and Zbtb38. Dnmt activity can also be modulated by factors such as Np95 (Uhrf1), a histone reader that stabilizes and potentiates Dnmt1  mediates chromatin compaction and gene silencing, and is actively regulated during neurogenesis  were specifically enriched in RGPs , and was directly activated by Tbr1.Among DNA demethylation genes, 2 Figure . Gadd45a2 Figure . Tet1 waMecp2, a methyl-cytosine reader linked to Rett syndrome , but not in any specific cell type, as ISH showed high levels in multiple zones. During embryonic neurogenesis, Mecp2 is necessary to limit Pax6 expression in Tbr2+ IPs, and to modulate the pace of PN maturation  by Pax6 and Tbr2 , GNAT , and Cbp/p300 (Crebbp/Ep300) families, regulate transcription and, in some cases, may also acetylate non-histone proteins. Some type-A HATs, such as p300, function as modular activating units that can be recruited by various EF/TF complexes, such as non-canonical PRC1-Auts2  function in cytoplasmic nucleosome biogenesis. Likewise, some class I HDACs (Hdac1/2) serve as modular repressive units, in complexes such as NuRD and Rest/CoRest.Histone lysine acetylation generally opens chromatin and activates transcription, while deacetylation represses transcription. Many families of histone acetyltransferases (HATs) and deacetylases (HDACs) mediate placement and reversal of the acetyl marks , an H3K14 acetyltransferase, is required for general transcriptional activation, especially in progenitor cells during embryonic development . Type-A HATs Cbp (Crebbp) and p300 (Ep300) were highly expressed in cortex, but without clear zonal specificity on ISH; nor were they directly regulated by Pax6, Tbr2, or Tbr1.The present analysis identified several HATs and HDACs with cell-type-specific expression, and extensive regulation by Pax6, Tbr2, and Tbr1 Figure . Among HKat2a and Kat6b, were specifically enriched in aIPs and bIPs in the VZ/SVZ  is required to prevent apoptosis , despite being an aIP and bIP marker , have thin cortex, especially layer 5, and reduced numbers of Tbr2+ IPs , and Brpf1.Two type-A HATs,  Figures . Kat2a  was specifically expressed in aIPs and bIPs . One function of Mitr (Hdac9) is to limit gene expression driven by Mef2 and physiological excitation , and thus indirectly potentiate Hdac5 expression. Hdac3 was moderately enriched in Tbr2-GFP+ cells, and widely expressed on ISH.Another HDAC, Hdac2 was enriched in Tbr2-GFP+ cells, and was expressed predominantly by differentiating neurons in the IZ/CP of cortex . In sum, Hdac1 and Hdac2 showed complementary enrichment in progenitors and neurons, respectively.Of the class I HDACs, x Figure , and othUhrf1, which recruits Dnmt1 and HATs to chromatin during proliferation  systems are considered antagonistic; genes marked with both H3K4me3 (activating) and H3K27me3 (repressive) are considered to be in a “bivalent” state, poised for long-term repression or activation. In mammals, the Trithorax and Polycomb systems have become more complex and diverse, with many tissue-specific isoforms and non-canonical subunits. While TrxG genes (as defined by PcG antagonism) also encompass other classes of molecules, such as chromatin remodelers  and demethylation. The best-known epigenetic systems using these marks are Trithorax/COMPASS complexes, which place H3K4 trimethyl (H3K4me3) and other marks at active promoters; and PRC2, which places repressive H3K27me3 marks that silence chromatin. The PRC2 system is furthermore connected to PRC1, which places another silencing histone mark—monoubiquitylation of H2A on K119 (H2AK119u1)—and functions synergistically with PRC2. In Wdr5, Rbbp5, Ash2l, Dpy30) and other subunits  and Lsd1 (Kdm1a)—both markers of neuronal differentiation beginning in progenitor zones , and was expressed at highest levels in CP  was also enriched in Tbr2-GFP+ cells (log2FC = +1.36), but was not detected on ISH. Likewise, Kmt2c (Mll3) was enriched in Tbr2-GFP+ cells (log2FC = +1.22), but not detected on ISH. Notably, Kmt2c was directly activated by Tbr1 (Supplementary Table Kmt2c (Mll3) is important for PN differentiation. Indeed, mutations in human KMT2C have been linked to intellectual disability  forms COMPASS-like complexes with Utx (Kdm6a), a demethylase that removes repressive H3K27me3 marks placed by PRC2  expression, Tbr1 may orchestrate not only the placement of activating H3K4me3 marks by Mll3, but also removal of repressive H3K27me3 marks by Utx.In the present analysis, both H3K4 methylase and demethylase genes were expressed predominantly in P Figure . Kmt2a (Kdm1a (Lsd1) was enriched in Tbr2-GFP+ cells (log2FC = +1.14), and was directly activated by Tbr2 (Supplementary Tables Rcor1/2), a repressor scaffold protein enriched in aIPs and bIPs , to promote a shift from multipolar to bipolar migration  expression, Tbr2 may drive this change of migration mode. Kdm1b  was similarly enriched in Tbr2-GFP+ cells (log2FC = +1.03), but its expression was not zone-specific on ISH , was enriched in neuronal lineages, and was directly activated by Tbr1  and Kdm5b (Jarid1b) respectively, to reconfigure the landscape of active promoters in differentiating PNs.Among H3K4 demethylases, H Figure . Another Figures . Thus, TKdm5b) is necessary to remove inappropriate H3K4me3 marks during development, and thereby deactivate neural progenitor genes such as Pax6 .Functionally, Jarid1b , another H3K4me3 demethylase, was expressed in a regional gradient  in the VZ/SVZ , so expression of Kdm5a could not be specifically assigned to RGPs or IPs.Z Figure . On micrAsh1l, an H3K36 methylase that may activate or repress transcription in different contexts , without direct regulation by Pax6, Tbr2, or Tbr1 , and Aebp2 or Jarid2. PRC2 also interacts with or is regulated by other EFs, such as Chd4  and Jmjd3 (Kdm6b).In mammals, a variety of PRC2 complexes with different subunit or isoform composition have been identified , a non-canonical PRC2 subunit that may inhibit PRC2 activity  was highly enriched in the neuronal lineage , which targets PRC2 to H3K36me3-marked chromatin, was expressed in a high rostral gradient in VZ/SVZ (counter to Suz12 and Eed). The Phf19 (PCL3) countergradient suggests that not only the abundance of PRC2 complexes, but also the formation of non-canonical PRC2 complexes, are regionally modulated within VZ/SVZ.Other canonical and non-canonical subunits of PRC2 also displayed cell-type-specific or regional expression patterns. s Figure , while Rs Figure . In conte Figure , and wasKdm6a; log2FC = +0.93) and Jmjd3  were both enriched in the Tbr2-GFP+ PN lineage, but ISH was not available for Kdm6a, and Kdm6b did not exhibit strict zonal expression  interacts with Tbr2 in IPs to derepress neuronal differentiation genes, such as Tbr1  was directly repressed by Pax6.H3K27me3 demethylases Utx  in addition to core subunits. Outside the proliferative zones, Suz12 and Eed are expressed very little, and PCL2 (Mtf2) is upregulated in neurons by Tbr1, leaving Ezh2 to potentially form non-PRC2 complexes  and Kdm6b (Jmjd3), which “unlock” chromatin by remove the H3K27me3 marks placed by PRC2. For regionalization, high canonical PRC2 activity is necessary in caudal VZ/SVZ for occipital cortex identity . Despite the important role of PRC2 in regionalization, the subunits with graded expression are not directly regulated by Pax6, Tbr2, or Tbr1  or Ring1b (Rnf2) E3 ligase; PcG ring finger (Pcgf) 2 or Pcgf4 (Bmi1); chromobox1-8 (Cbx1-8); Hph1-3 (Phc1-3); and substoichiometric amounts of Scm (Scmh1/2) , and drives chromatin compaction , a type-A HAT, to activate  transcription.Non-canonical PRC1 complexes contain Rybp or Yaf2 instead of Cbx, and may contain canonical (Pcgf2/4) or non-canonical (Pcgf1/3/5/6) Pcgf proteins -deficient RGPs, neurogenesis is prolonged  appeared to be the predominant E3 ligase in developing neocortex. Rnf2 was enriched in Tbr2-GFP+ cells, and was seen in all zones by ISH, though highest in the VZ  was barely detectable on microarrays and ISH.In the present analysis, Z Figure . In contBmi1  was moderately enriched in Tbr2-GFP+ cells, and more highly expressed than Pcgf2. Multiple Cbx genes were expressed in developing neocortex, but none exhibited cell-type specificity. However, Cbx4 was directly activated by Pax6. Since Cbx4 promotes sumoylation of Dnmt3a  in progenitors, to alpha-1 in neurons (Csnk2a1). Rybp was highly enriched in aIPs and bIPs (log2FC = +0.81), and was expressed at lower levels in neurons  implicated in brain development , and are minimally regulated by Pax6→ Tbr2→ Tbr1. In contrast, non-canonical PRC1 complexes exhibit differentiation-related changes, such as upregulation of Kdm4c (Jmjd2c), which encodes an enhancer-associated H3K9 demethylase and scaffold that primes cells for differentiation  was enriched in Tbr2-GFP+ lineages (log2FC = +0.55), but was repressed by Tbr2 , a histone H3K9ac reader, was enriched in neurons of the IZ and CP  or Brg1 (Smarca4) in BAF  or Snf2l (Smarca1) in ACF/CHRAC and NuRF types of ISWI complexes, respectively  in INO80 complexes , ISWI (Imitation Switch), CHD (chromodomain helicase DNA-binding), and INO80 (inositol auxotrophy 80). The complexes are defined by their ATPase subunits: Brm , and 15 in INO80 complexes  are also implicated in epigenetic regulation of neurodevelopment  mutant mice exhibit excessive, prolonged proliferation of cortical progenitors, especially IPs  mutant mice exhibit reduced proliferation, at least in cerebellum  was expressed in all zones of developing neocortex, with highest levels in VZ/SVZ . Smarca1 (Snf2l) was expressed in multiple zones, and did not show differential expression on Tbr2-GFP microarray. Thus, both ISWI ATPases were widely expressed in developing neocortex, although Smarca5 (Snf2h) was somewhat higher in progenitors. This interpretation matches a previous description  complexes, was specifically enriched in RGPs , but also either RbAP48 (Rbbp4) or RbAP46 (Rbbp7) . These data suggest that NuRF complexes are restricted to RGPs, and are comprised of Bptf/Snf2l/RbAP46 , a component of NoRC (nucleolar remodeling complex) in the Snf2h-containing ACF/CHRAC group of ISWI remodelers, was also highly enriched in IPs  was expressed at high levels in VZ, and was moderately enriched in the Tbr2-GFP+ lineage (log2FC = +0.85); thus, WICH complexes (Wstf/Snf2h) may be enriched in progenitors, especially IPs.s Figure . SimilarBaz2b by Tbr2 and Tbr1 suggests that downregulation of some ISWI complexes (possibly a Baz2b-containing NoRC variant) is important for differentiation from IPs to PNs.Overall, the present analysis suggests that NuRF complexes are specifically present in RGPs, while NoRC complexes are particularly abundant in IPs Figure . The dirIno80 was detected primarily in VZ, but was not enriched in Tbr2-GFP− or GFP+ lineages (Supplementary Table Ino80b (Ies2), which activates the ATPase activity of Ino80, was specifically expressed in RGPs (log2FC = −0.45), suggesting that Ino80-containing complexes are enriched and activated in RGPs. The INO80 remodelers are important in DNA replication and repair, as well as transcriptional regulation  were detected in multiple zones, and were moderately enriched in Tbr2-GFP+ cells . Most Srcap complex subunits were widely expressed, while several p400 complex subunits, such as Kat5 (Tip60), were relatively enriched in neurons. Pax6, Tbr2, and Tbr1 were not implicated in the regulation of INO80 complex subunits.Together, these findings suggest that Ino80-containing complexes are specifically active in RGPs, while p400/Tip60 complexes are most active in postmigratory CP neurons. The functions of INO80 complexes in cortical development are unknown.Chd ATPase genes, only Chd7 exhibited cell-type or region-specific expression—indeed, both. Chd7 was enriched in Tbr2-GFP+ cells (log2FC = +0.80) on microarray, and was expressed specifically in VZ on ISH, identifying Chd7 as a specific marker of aIPs. Within VZ, Chd7 exhibited high caudal expression  and globally activates transcription . NuRD complexes have at least six subunits, each of which has multiple alternatives or isoforms: Chd3-5, Hdac1/2; Mbd2/3; Mta1-3; Gatad2a/b; and Rbbp4/7  enriched in Tbr2-GFP+ cells on microarray , and was directly repressed by Tbr2 . Hdac1 was expressed in all zones but enriched in VZ/SVZ, while Hdac2 was moderately enriched in neurons , cause Coffin-Siris syndrome, a microcephaly disorder with intellectual disability  or Brg1 (Smarca4); Baf155/170 (Smarcc1/2); Baf60a-c (Smarcd1-3); Baf53a/b (Actl6a/b); and Baf47 (Smarcb1). In addition, mammalian BAF complexes contain ≥10 other subunits, such as Baf250a/b (Arid1a/b) , Ss18, and Baf45a/d (Phf10/Dpf2); in neuronal BAF (nBAF), these subunits are replaced with Baf53b (Actl6b), Crest (Ss18l1), and Baf45b/c (Dpf1/3), respectively. Interestingly, the shift from Baf53a (Actl6a) to Baf53b (Actl6b) is driven by microRNA (miR)-9* and miR-124, which target Actl6a (Baf53a) for degradation  is directly repressed by Tbr2 and Tbr1.In cortical development, BAF has been shown to exchange four subunits during differentiation from progenitors to neurons  was ubiquitous, but Smarca2 (Brm) was specifically expressed by postmigratory PNs  was ubiquitously expressed, while Smarcd3 (Baf60c) was enriched in the CP  was ubiquitously expressed, while Smarcc2 (Baf170) was abundant in CP (Supplementary Table Smarcc2) has been linked to repression of IP genesis and neurogenesis  to Actl6b (Baf53b) was confirmed on ISH  suggested that Actl6a was expressed in not only RGPs, but also IPs. Likewise, npBAF subunits Phf10 (Baf45a) and Dpf2 (Baf45d) were highly expressed in VZ/SVZ  were highly expressed in IZ/CP. Upregulation of Dpf3 (Baf45c) in differentiating neurons was directly activated by Tbr1 and Tbr2.The previously described  was specifically expressed in RGPs (log2FC = −1.07), while Bcl7a (Baf40a) was enriched in IPs and neurons (log2FC = +1.32). These findings define Baf40c and Baf40a as components of npBAF and nBAF, respectively  was ubiquitously expressed, while Arid1b (Baf250b) was enriched in the CP  and BAF250b-containing (BAF-B) complexes -containing BAF: Baf180 (Pbrm1), Baf200 (Arid2), Baf45a (Phf10), and Brd7  in PNs , is formed by the incorporation of four specific subunits in Brg1  and Ctip2/Baf100b (Bcl11b) are BAF subunit TFs with major roles in PN differentiation and regionalization  and Bcl11b (log2FC = +1.75) were highly enriched in the Tbr2-GFP+ lineage, and both were expressed predominantly in neuronal differentiation zones. Additionally, Bcl11a was expressed in a high caudal gradient, as described  and Jmjd3 (Kdm6b), and potentiate their H3K27me3 demethylase activity , to silence neuronal genes  have been distinguished, and other CoRest interactions and functions have been discovered  to prevent neuronal differentiation , while Rcor2 was expressed mainly in SVZ/IZ and inner VZ , together with its bilaminar expression pattern in VZ and SVZ  was ubiquitously expressed . Also, Kdm1a (Lsd1) was directly bound and activated byTbr2 . In contrast to Kdm1a (Lsd1), which was activated by Tbr2, Insm1 was repressed by both Tbr2 and Pax6  ncRNA (lncRNA) that binds chromatin to mediate X-inactivation   to promote BAF subunit switching, and are themselves repressed by Rest , 9630028B13Rik (log2FC n.a.), and A930024E05Rik (log2FC = +1.97) , was similarly expressed in SVZ/IZ, consistent with bIPs and new PNs , a lncRNA expressed specifically in bIPs with a high lateral gradient , and was localized in the inner VZ  was highly expressed in new neurons of the IZ and CP  of basal IPs with proportionately decreased Tbr2 expression  was directly repressed by Tbr2  is a MYST family HAT that activates gene expression, and is required for forebrain growth  deficient embryos, but deficiency of the MYST coactivator, Brpf1, has been found to reduce IP genesis and cortical growth  and TF  genes in IPs , a YEATS domain acetylation reader that directly mediates Tbr1 repression for upper layer identity  to promote PN differentiation  to control laminar identity , a MYST family HAT that promotes layer 5 differentiation, as well as cortical growth  was associated with increased abundance of layer 5 neurons  in layer 5 differentiation is supported by the phenotype of Brpf1 mutant mice: Brpf1 is an activator of Morf (Kat6b), and Brpf1 mutants have prominent layer 5 defects  and Bcl11a (Ctip1), in CP and IZ/CP respectively  in PNs (Figure The present study demonstrates that many types of EFs are direct targets of gene activation or repression by Pax6, Tbr2, or Tbr1 Table . In anots Figure . Overalls Figure . The Paxs Figure .Epigenetic mechanisms appear well-suited to regulation of regional and laminar identity, persistent phenotypes that are initially determined in progenitor cells, then propagated into IPs and finally, new PNs. For example, the cortical “protomap” is initially specified in RGPs, then propagated into IPs and PNs, where regional identity continues to be refined (Bedogni et al., Besides EFs, other target genes regulated by Pax6, Tbr2, and Tbr1 can be identified using the same approach, and are currently under analysis. Through these studies, it will be possible to comprehensively profile gene expression by RGPs, IPs, and PNs; and to better understand how Pax6, Tbr2, and Tbr1 control the genesis of cortical PNs.GE designed and conducted experiments, produced new microarray data from Tbr1/2 mutant embryos, analyzed results, and wrote the manuscript. FB and RH produced microarray data. TB and JM analyzed data from microarray and ChIP-seq experiments. SL and JR produced and analyzed Pax6 ChIP-seq data. RH designed experiments, analyzed data, and wrote the manuscript.JR is cofounder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.