Tissue-specific Transcription Factors Research Articles

On epigenetic stochasticity, entropy and cancer risk.

Epigenetic changes are known to accrue in normal cells as a result of ageing and cumulative exposure to cancer risk factors. Increasing evidence points towards age-related epigenetic changes being acquired in a quasi-stochastic manner, and that they may play a causal role in cancer development. Here, I describe the quasi-stochastic nature of DNA methylation (DNAm) changes in ageing cells as well as in normal cells at risk of neoplastic transformation, discussing the implications of this stochasticity for developing cancer risk prediction strategies, and in particular, how it may require a conceptual paradigm shift in how we select cancer risk markers. I also describe the mounting evidence that a significant proportion of DNAm changes in ageing and cancer development are related to cell proliferation, reflecting tissue-turnover and the opportunity this offers for predicting cancer risk via the development of epigenetic mitotic-like clocks. Finally, I describe how age-associated DNAm changes may be causally implicated in cancer development via an irreversible suppression of tissue-specific transcription factors that increases epigenetic and transcriptomic entropy, promoting a more plastic yet aberrant cancer stem-cell state. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Tissue-specific transcriptional programming of macrophages controls the microRNA transcriptome targeting multiple functional pathways

Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6-deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization, and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, -223, and -203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Participation of the TBP-associatedfactors (TAFs) in cell differentiation.

The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associatedfactors (TAFs)during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs'regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Ncrna-a3 Epigenetically Regulates TAL1 and Its Target Genes to Drive Erythroid Differentiation

Erythropoiesis is the development of enucleated red blood cells from multipotent hematopoietic stem cells. This complex process is tightly regulated by the dynamic changes in occupancy of tissue specific transcription factors (TFs) that activate and repress genes appropriately. TAL1, a critical regulator is expressed throughout the development of mature erythroblasts from multi-progenitors. Loss of TAL1 leads to defective erythropoiesis. As opposed to TAL1's role in erythropoiesis, itis an extraneous factor in developing lymphoid cells and aberrant expression of TAL1 in T-cells is associated with T-ALL. The differential regulation during normal and malignant hematopoiesis remains largely unclear. We found that a distil RNA - ncRNA-a3, is transcribed from erythroid specific enhancer, 50kbp downstream of the TAL1 TSS. We hypothesized that ncRNA-a3 plays an important role in regulating TAL1 and its target gene transcription in an erythroid specific manner. To test this hypothesis, we examine the regulatory potential of ncRNA-a3 towards the activation of TAL1 expression in the context of hematopoiesis and leukemogenesis. We used RNA interference to deplete ncRNA-a3 expression across several hematopoietic and leukemic cells. The decrease in ncRNA-a3 resulted in significant reduction of TAL1 transcript and protein levels in hematopoietic cells while TAL1 transcripts showed little or no reduction in leukemic cells. Subsequently, transcriptomic profile changes in erythroleukemic K562 cells showed downregulation of erythroid specific targets of TAL1. To ascertain the role of ncRNA-a3 in erythropoiesis, we used siRNA against ncRNA-a3 in primary CD34+ cells and subjected cells to differentiation. Quantification of early and late erythroblasts by colony forming assay and flow cytometry indicated impaired erythroid differentiation and maturation in orthochromatic and polychromatic stages. Further, RNA-seq analysis of ncRNA-a3 knockdown cells revealed defects in globin gene function, heme biosynthesis and nucleosome condensation compared to control CD34+ cells during time course EPO induced erythroid differentiation. Mechanistically, we observed ncRNA-a3s occupancy at TAL1 regulatory regions from ChIRP-qPCR across TAL1 locus. NG Capture-C data depicting changes in interaction frequencies indicated that ncRNA-a3 maintains canonical interactions at the TAL1 locus. Knockdown of ncRNA-a3 negatively impacted the chromatin accessibility in TAL1 and TAL1 target loci. Perturbation of chromatin landscape affected TAL1 and TAL1 binding partner - P300 and BRG1 occupancy at TAL1 target sites. To distinguish reduced TAL1 binding as a consequence of lower levels of TAL1, from RNA dependent binding of TAL1, we examined TAL1, P300 and BRG1 chromatin occupancy through Cut&Run in conjunction with RNase treatment. Effective depletion of global RNAs resulted in reduced enrichment of TAL1, P300 and BRG1 bound fragments indicating possible RNA dependent binding of TAL1 and its cofactors. TAL1 coimmunoprecipitation using RNase treatment (1) and ncRNA-a3 KD (2), demonstrated that TAL1s interaction with P300 and BRG1 is RNA dependent, but ncRNA-a3 independent. RNA-IP results further confirmed that ncRNA-a3 is associated with TAL1 through its interacting epigenetic cofactors P300 and BRG1. Together, our data indicate that although RNA dependent binding of TAL1 and its epigenetic cofactors is cell type or target locus specific, ncRNA-a3 modulates the occupancy of TAL1, P300 and BRG1 at the TAL1 locus during erythroid specific TAL1 transcription.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

Identification of a Novel Gene Regulatory Element in Human Erythroid Progenitor Cells

Early erythropoiesis involves commitment of multi-lineage progenitors to erythroid progenitor cells and differentiation into erythroid burst-forming unit (BFU-E) cells, then erythroid colony-forming unit (CFU-E) cells. In contrast to terminal erythroid differentiation, regulation of gene expression in early erythropoiesis is poorly defined. To address this knowledge gap, we identified active enhancers in erythroid progenitor cells linked to known disease genes and genes associated with erythrocyte traits in genome wide association studies. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from mobilized peripheral blood were cultured towards the erythroid lineage. From these cultures, FACS-based methods were utilized to purify four stages of erythroid progenitor cells, termed EP1 to EP4, and five populations of terminally differentiated erythroblasts, from proerythroblasts to orthochromatic erythroblasts. ChIP-seq was performed using antibodies to H3K4me1, H3K27Ac, H3K27me3, and H3K9me2/3. Chromatin accessibility was determined by ATAC-seq. Patterns of gene expression were determined by RNA-seq. Active enhancers were identified using the ModHMM algorithm. Numerous erythroid progenitor cell-specific active enhancers were identified near genes linked to known disease genes and GWAS-associated erythrocyte traits. rs162375, a single nucleotide polymorphism (SNP) linked to mean corpuscular hemoglobin, was in a 475bp Long Terminal Repeat Retrotransposon (LTR) element upstream of the RRP1B gene. The LTR enhancer near the RRP1B gene is a member of the LTR15 family. Transposable elements are a source of novelty in primate gene regulation when they acquire binding sites for critical tissue-specific transcription factors, and then these tissue-specific regulatory regions direct tissue-specific expression of nearby genes. The SNP-containing LTR15 erythroid progenitor-specific enhancer upstream of RRP1B contains 2 conserved GATA motifs and a conserved RUNX motif. The enhancer region had a peak of open chromatin determined by ATAC-seq, and H3K27 acetylation and strong GATA1 binding determined by ChIP-seq. These findings were present only in erythroid progenitor cells, not in earlier hematopoietic cells, terminally differentiating erythroid cells, or other non-erythroid cells. CRISPR-based gene editing was utilized to delete the LTR15 enhancer in CD34+ HSPCs cultured towards the erythroid lineage into erythroid progenitor cells. mRNA expression of RRP1B was decreased to 70.2% of wild type. Luciferase-based reporter gene studies demonstrated the enhancer increased expression of a minimal promoter 3.2-fold over background. Mutation of the GATA and RUNX sites individually or together decreased reporter gene activity to background levels. Bioinformatic analyses identified 291 additional erythroid progenitor cell specific LTR15 element active enhancers, with most containing two GATA motifs and a RUNX motif. The majority of the novel enhancer regions were also marked by progenitor cell-specific ATAC peaks and strong GATA1 binding in ChIP-seq. GATA1 and RUNX, both present in early erythropoiesis, are required to activate the regulatory elements in erythroid progenitor cells. Decreasing levels of RUNX in terminal erythroid differentiation parallels loss of enhancer activity. Since LTR15 first appeared in Catarrhine, old-world monkeys, these data indicate this novel regulatory element has recently evolved to contribute to regulation of gene expression in early erythropoiesis in primates.

SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs.

During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.

Differentiation, regulation and function of regulatory T cells in non-lymphoid tissues and tumors.

Regulatory T cells (Tregs) play a substantial role in inhibiting excessive immune response. A large number of studies have focused on the tissue homeostasis maintenance and remodeling characteristics of Tregs in non-lymphoid tissues, such as the skin, colon, lung, brain, muscle, and adipose tissues. Herein, we overview the kinetics of Treg migration to non-lymphoid tissues and adaptation to the specific tissue microenvironment through the development of tissue-specific chemokine receptors, transcription factors, and phenotypes. Additionally, tumor-infiltrating Tregs (Ti-Tregs) play an important role in tumor generation and immunotherapy resistance. The phenotypes of Ti-Tregs are related to the histological location of the tumor and there is a large overlap between the transcripts of Ti-Tregs and those of tissue-specific Tregs. We recapitulate the molecular underpinnings of tissue-specific Tregs, which might shed new light on Treg-based therapeutic targets and biomarkers for inflammatory diseases and cancer.

Tissue-Specific Regulation of CFTR Gene Expression.

More than 2000 variations are described within the CFTR (Cystic Fibrosis Transmembrane Regulator) gene and related to large clinical issues from cystic fibrosis to mono-organ diseases. Although these CFTR-associated diseases have been well documented, a large phenotype spectrum is observed and correlations between phenotypes and genotypes are still not well established. To address this issue, we present several regulatory elements that can modulate CFTR gene expression in a tissue-specific manner. Among them, cis-regulatory elements act through chromatin loopings and take part in three-dimensional structured organization. With tissue-specific transcription factors, they form chromatin modules and can regulate gene expression. Alterations of specific regulations can impact and modulate disease expressions. Understanding all those mechanisms highlights the need to expand research outside the gene to enhance our knowledge.

Dissecting small cell lung cancer subtypes with cell-free DNA fragmentomes.

8592 Background: Although small cell lung cancer (SCLC) is managed as a single cancer type, new evidence supports that subtypes (high vs low neuroendocrine) of SCLC acquire diverse transcriptional and epigenetic states. Furthermore, distinct SCLC subtypes acquire unique therapeutic vulnerabilities, such as the low-neuroendocrine highly inflamed SCLC subtype that has been linked to immunotherapy sensitivity. We have previously shown that DELFI (DNA evaluation of fragments for early interception) can be used to non-invasively distinguish SCLC from non-small cell lung cancer (NSCLC). Here, we report a preliminary analysis of using the DELFI approach for distinguishing among SCLC subtypes. Methods: Circulating cell-free DNA (cfDNA) was isolated from plasma samples of patients diagnosed with relapsed SCLC and treated with durvalumab plus olaparib in a phase II trial (NCT02484404). Over 200 cfDNA libraries were prepared for whole genome sequencing in batches with internal controls. To infer tumor gene expression profiles in cfDNA, we investigated genome-wide signals of tissue-specific transcription factors differentially regulated in SCLC, and applied a novel DELFI-based approach to inform SCLC molecular subtypes. Clinical information, patient-matched tissue transcriptome (RNA-seq), and chromatin accessibility data (ATAC-seq) were examined in orthogonal analyses. Results: The DELFI classifier detected SCLC cases (N = 47) with a median DELFI score of 1.0 (95% CI 0.99-1), significantly higher than previously reported scores for other lung cancer subtypes. Comparison of DELFI fragmentome signals to publicly available tumor transcriptomes shows subtype-level concordance ( r = 0.78, p < 0.001, Pearson), particularly in pre-treatment SCLC cases separating high- vs low- neuroendocrine subtypes. High-neuroendocrine SCLCs exhibited a decrease in aggregate cfDNA fragment coverage at ASCL1 transcription factor binding sites relative to low-neuroendocrine SCLCs (r = 0.90, p < 0.001, Pearson). Additionally, low-neuroendocrine SCLCs revealed a significant decrease in aggregate cfDNA fragment coverage at genomic binding sites regulated by hematopoietic transcription factors, reflecting the inflamed phenotype of this SCLC subtype. Integration of these approaches provided a cfDNA fragmentation-based machine learning model that distinguished SCLC subtypes with high performance. Conclusions: Genome-wide cfDNA fragmentome analyses can differentiate high- and low-neuroendocrine SCLC subtypes. Given the challenges for performing SCLC biopsies in a clinical setting, we believe this approach could be a viable method of subtyping SCLC in a non-invasive manner.

Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes

Transcriptional regulation is commonly governed by alternative promoters. However, the regulatory architecture in alternative and reference promoters, and how they differ, remains elusive. In 100 CAGE-seq libraries from hepatocellular carcinoma patients, here we annotate 4083 alternative promoters in 2926 multi-promoter genes, which are largely undetected in normal livers. These genes are enriched in oncogenic processes and predominantly show association with overall survival. Alternative promoters are narrow nucleosome depleted regions, CpG island depleted, and enriched for tissue-specific transcription factors. Globally tumors lose DNA methylation. We show hierarchical retention of intragenic DNA methylation with CG-poor regions rapidly losing methylation, while CG-rich regions retain it, a process mediated by differential SETD2, H3K36me3, DNMT3B, and TET1 binding. This mechanism is validated in SETD2 knockdown cells and SETD2-mutated patients. Selective DNA methylation loss in CG-poor regions makes the chromatin accessible for alternative transcription. We show alternative promoters can control tumor transcriptomes and their regulatory architecture.

Hematopoietic/erythroid enhancers activate nearby target genes by extending histone H3K27ac and transcribing intergenic RNA.

Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.

Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells.

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.

The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration.

Tissue-specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.

General transcription factor TAF4 antagonizes epigenetic silencing by Polycomb to maintain intestine stem cell functions

Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.

Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.

An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.

Abstract B026: Implicating SATB2 loss in a metaplasia-dysplasia sequence in human inflammatory bowel disease

Abstract Epithelial metaplasia, or the histologically evident replacement of one differentiated somatic cell type with another, offers insights into mucosal homeostasis as an adaptation to diverse inflammatory pathogenic stimuli. The cellular framework for metaplasia can be created by coordination of tissue-specific transcription factors (TFs) with epigenetic regulators of chromatin remodeling, allowing altered TF binding of inflammation-associated exposed genomic loci. For example, intestinal metaplasia (aberrant goblet cell differentiation and CDX2 expression, a pan-intestinal TF) is a common metaplastic response to chronic injury in the upper gastrointestinal tract, associated with increased risk of epithelial dysplasia and cancer. Inflammatory bowel disease (IBD) is an etiologically complex chronic inflammatory disease characterized by relapsing cycles of intestinal injury and healing. IBD patients carry increased risk of colitis-associated dysplasia and colorectal cancer (CAC) proportional to disease duration, extent, and severity. SATB2, or special AT-rich binding protein 2, is a chromatin organizing protein with homeostatic roles in osteoblastic, neural, and colonic differentiation. Intriguingly, others have recently shown that SATB2 is uniquely lost in human IBD dysplasia and CAC, in contrast to sporadic CRC where its expression is typically retained. (By contrast, CDX2 shows retained expression in both CAC and CRC.) Other recent work has shown that colon-specific Satb2 knockout in mice results in conversion of colon to small intestine, including emergence of villi and Paneth cells. Colonic mucosal biopsies from chronic IBD patients show histologic features reminiscent of small intestine with so-called Paneth cell metaplasia (PCM) and villiform, distorted growth. The molecular mediators of this epithelial remodeling in IBD are unknown. We hypothesized that SATB2 loss could result in a form of “small intestinal metaplasia” in human IBD colon. To investigate this, we selected a cohort of 20 IBD distal colon mucosal biopsies with marked crypt architectural changes and PCM and performed SATB2 immunostaining (n=7 Crohn’s disease, n=13 ulcerative colitis). Indeed, 19 of 20 such samples showed striking evidence of SATB2 loss. The pattern of SATB2 loss was heterogeneous; some tissue fragments showed complete epithelial loss as expected in small intestine, whereas others showed patchy loss in alternating crypts, even some showing heterogeneous expression within the same crypt. As positive and negative controls, we included normal distal colon and terminal ileum samples, observing expected intact and negative SATB2 expression, respectively. Of note, CDX2 was strongly expressed across the entire cohort, even in IBD tissue with loss of SATB2 expression. We propose that SATB2 loss is a pre-neoplastic metaplastic response to chronic injury in human colon. Future work will investigate how SATB2 loss enables a plasticity state permissive for emergence of TP53 mutations, dysplasia, and CAC. Citation Format: Maged Zeineldin, Reem Abu-Shamma, Tatianna Larman. Implicating SATB2 loss in a metaplasia-dysplasia sequence in human inflammatory bowel disease [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B026.

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.