Repression Of Transcription Factors Research Articles

DcWRKY15 positively regulates anthocyanin biosynthesis during petal coloration in Dianthus caryophyllus

Petal pigmentation in carnations is closely associated with the biosynthesis of anthocyanins. This biosynthetic process is tightly regulated by transcription factors, which activate or repress key genes involved in anthocyanin production. Here, we aim to explore the mechanisms involved in the transcriptional regulation of anthocyanin biosynthesis in carnation petals. We identified DcWRKY15 as a critical regulator influencing anthocyanin production in these petals. DcWRKY15 expression showed a strong correlation with the expression levels of genes associated with anthocyanin biosynthesis, peaking during early petal development stages. The findings from the dual-LUC, VIGS, Y1H and EMSA assays demonstrated that DcWRKY15 played a positive regulatory role in anthocyanin biosynthesis. DcWRKY15 achieved this by binding directly to the promoters of DcCHS and DcF3H, thereby enhancing their expression. Additionally, DcWRKY15 interacts with the repressor DcMYB2, which reduces its capacity to enhance anthocyanin biosynthesis, particularly during the later stages of petal development. These findings offer new insights into the molecular mechanisms responsible for petal coloration in carnations, highlighting the complex interplay between activator and repressor transcription factors.

Uncovering the whole genome silencers of human cells via Ss-STARR-seq

Silencers, the yin to enhancers’ yang, play a pivotal role in fine-tuning gene expression throughout the genome. However, despite their recognized importance, comprehensive identification of these regulatory elements in the genome is still in its early stages. We developed a method called Ss-STARR-seq to directly determine the activity of silencers in the whole genome. In this study, we applied Ss-STARR-seq to human cell lines K562, LNCaP, and 293 T, and identified 134,171, 137,753, and 125,307 silencers on a genome-wide scale, respectively, these silencers function in various cells in a cell-specific manner. Silencers exhibited a substantial enrichment of transcriptional-inhibitory motifs, including REST, and demonstrated overlap with the binding sites of repressor transcription factors within the endogenous environment. Interestingly, H3K27me3 did not reflect silencer activity but facilitated the silencer’s inhibitory role on gene expression. Additionally, the silencer did not have any significant histone markers at the genome-wide level. Our findings unveil that aspect-silencers not only transition into enhancers throughout diverse cell lines but also achieve functional conversion with insulators. Regarding to biological effects, knockout experiments underscored the functional redundancy and specificity of silencers in regulating gene expression and cell proliferation. In summary, this study pioneers the elucidation of the genome-wide silencer landscape in human cells, delineates their global regulatory features, and identifies specific silencers influencing cancer cell proliferation.

The Unexplored Role of Connexin Hemichannels in Promoting Facioscapulohumeral Muscular Dystrophy Progression.

DUX4 is typically a repressed transcription factor, but its aberrant activation in Facioscapulohumeral Muscular Dystrophy (FSHD) leads to cell death by disrupting muscle homeostasis. This disruption affects crucial processes such as myogenesis, sarcolemma integrity, gene regulation, oxidative stress, immune response, and many other biological pathways. Notably, these disrupted processes have been associated, in other pathological contexts, with the presence of connexin (Cx) hemichannels-transmembrane structures that mediate communication between the intracellular and extracellular environments. Thus, hemichannels have been implicated in skeletal muscle atrophy, as observed in human biopsies and animal models of Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, and Dysferlinopathies, suggesting a potentially shared mechanism of muscle atrophy that has not yet been explored in FSHD. Despite various therapeutic strategies proposed to manage FSHD, no treatment or cure is currently available. This review summarizes the current understanding of the mechanisms underlying FSHD progression, with a focus on hormones, inflammation, reactive oxygen species (ROS), and mitochondrial function. Additionally, it explores the potential of targeting hemichannels as a therapeutic strategy to slow disease progression by preventing the spread of pathogenic factors between muscle cells.

PerR functions as a redox-sensing transcription factor regulating metal homeostasis in the thermoacidophilic archaeon Saccharolobus islandicus REY15A.

Thermoacidophilic archaea thrive in environments with high temperatures and low pH where cells are prone to severe oxidative stress due to elevated levels of reactive oxygen species (ROS). While the oxidative stress responses have been extensively studied in bacteria and eukaryotes, the mechanisms in archaea remain largely unexplored. Here, using a multidisciplinary approach, we reveal that SisPerR, the homolog of bacterial PerR in Saccharolobus islandicus REY15A, is responsible for ROS response of transcriptional regulation. We show that with H2O2 treatment and sisperR deletion, expression of genes encoding proteins predicted to be involved in cellular metal ion homeostasis regulation, Dps, NirD, VIT1/CCC1 and MntH, is significantly upregulated, while expression of ROS-scavenging enzymes remains unaffected. Conversely, the expression of these genes is repressed when SisPerR is overexpressed. Notably, the genes coding for Dps, NirD and MntH are direct targets of SisPerR. Moreover, we identified three novel residues critical for ferrous ion binding and one novel residue for zinc ion binding. In summary, this study has established that SisPerR is a repressive redox-sensing transcription factor regulating intracellular metal ion homeostasis in Sa. islandicus for oxidative stress defense. These findings have shed new light on our understanding of microbial adaptation to extreme environmental conditions.

Histone Modification-Dependent Transcriptional Regulation of Defence Genes in Early Response of Arabidopsis to Spodoptera litura Attack.

Histone modification is a cellular process for transcriptional regulation. In herbivore-damaged plants, activation of genes involved in defence responses is required for antiherbivore properties, but little is known about how the chromatin remodelling system is involved. In Arabidopsis (Arabidopsis thaliana) plants responding to Spodoptera litura larvae, HAC1 and HDA6, a histone acetyltransferase and a histone deacetylase, respectively, were found here to be involved in histone H3 (Lys9; H3K9) acetylation/deacetylation at the promoter region of the plant defensin gene PDF1.2 and the gene body of ethylene response factor 13 (ERF13) as early as 2 h after the onset of herbivore attack. The H3K9 acetylation was responsible for the robust upregulation of PDF1.2 later, at 24 h, and ERF13 even earlier, at 1 h. TOPLESS (TPL) and TOPLESS-related (TPR) corepressors interacted with HDA6 to deacetylate H3K9 at PDF1.2 and ERF13, while negatively regulating the expression of PDF1.2 but not ERF13. Furthermore, TPL also interacted with ERF13, resulting in ERF13-mediated regulation of PDF1.2. Taken together, these data suggest a model of promoter-restricted, TPL/TPR-directed histone deacetylation and transcription factor repression in healthyArabidopsis plants for the feedback regulation of the antiherbivore response.

Epistasis between genetic variations on MdMYB109 and MdHXK1 exerts a large effect on sugar content in apple fruit.

Many quantitative traits are controlled by multiple genetic variations with minor effects, making it challenging to resolve the underlying genetic network and to apply functional markers in breeding. Affected by up to a hundred quantitative trait loci (QTLs), fruit-soluble sugar content is one of the most complex quantitative traits in apple (Malus sp.). Here, QTLs for apple fruit sucrose and fructose content were identified via QTL mapping and bulked-segregant analysis sequencing (BSA-seq) using a population derived from a 'Jonathan' × 'Golden Delicious' cross. Allelic variations and non-allelic interactions were validated in the candidate genes within these defined QTL regions. Three single-nucleotide polymorphisms (SNPs) (SNP -326 C/T, SNP -705 A/G, and SNP -706 G/T) in the MdMYB109 promoter region affected the binding ability of the repressive transcription factor MdWRKY33, leading to increased MdMYB109 expression. MdMYB109 bound directly to the promoter of the sucrose transporter gene MdSUT2.2 and activated its expression, raising fruit sucrose content. A SNP (SNP1060 A/G) in the hexokinase gene MdHXK1 affected the phosphorylation of the transcription factor MdbHLH3, and phosphorylated MdbHLH3 interacted with MdMYB109 to co-activate MdSUT2.2 expression and increase fruit sucrose content. Adding the joint effects of the genotype combinations at the SNP markers based on the SNPs in MdMYB109 and MdHXK1 increased the prediction accuracy of a genomics-assisted prediction (GAP) model for total soluble solid content from 0.3758 to 0.5531. These results uncovered functional variations in MdMYB109 and MdHXK1 regulating apple fruit sucrose content. The updated GAP model with improved predictability can be used efficiently in apple breeding.

Abundant repressor binding sites in human enhancers are associated with the fine-tuning of gene regulation.

The regulation of gene expression relies on the coordinated action of transcription factors (TFs) at enhancers, including both activator and repressor TFs. We employed deep learning (DL) to dissect HepG2 enhancers into positive (PAR), negative (NAR), and neutral activity regions. Sharpr-MPRA and STARR-seq highlight the dichotomy impact of NARs and PARs on modulating and catalyzing the activity of enhancers, respectively. Approximately 22% of HepG2 enhancers, termed "repressive impact enhancers" (RIEs), are predominantly populated by NARs and transcriptional repression motifs. Genes flanking RIEs exhibit a stage-specific decline in expression during late development, suggesting RIEs' role in trimming enhancer activities. About 16.7% of human NARs emerge from neutral rhesus macaque DNA. This gain of repressor binding sites in RIEs is associated with a 30% decrease in the average expression of flanking genes in humans compared to rhesus macaque. Our work reveals modulated enhancer activity and adaptable gene regulation through the evolutionary dynamics of TF binding sites.

The function of the ZFP189 transcription factor in the nucleus accumbens facilitates cocaine-specific transcriptional and behavioral adaptations.

Distinguishing the brain mechanisms affected by distinct addictive drugs may inform targeted therapies against specific substance use disorders (SUDs). Here, we explore the function of a drug-associated, transcriptionally repressive transcription factor (TF), ZFP189, whose expression in the nucleus accumbens (NAc) facilitates cocaine-induced molecular and behavioral adaptations. To uncover the necessity of ZFP189-mediated transcriptional control in driving cocaine-induced behaviors, we created synthetic ZFP189 TFs of distinct transcriptional function, including ZFP189VPR, which activates the expression of target genes and exerts opposite transcriptional control to the endogenously repressive ZFP189. By virally delivering synthetic ZFP189 TFs to the NAc of mice, we discover that the transcriptional control exerted by synthetic or endogenous ZFP189 solely alters behavioral adaptations to cocaine but not morphine, saline, or sucrose. Further, these synthetic ZFP189 TFs are only capable of producing gene-expression changes in rodents exposed to cocaine, but not morphine or saline. In these cocaine exposed mice, the gene-expression profile produced by ZFP189VPR is inversely related to the cocaine-induced transcriptional response, as characterized by Upstream Regulator Analysis in Ingenuity Pathway Analysis. Lastly, we demonstrate that NAc ZFP189WT increases vulnerability to cocaine reinforcement through selective sensitization to the reinforcing effects of small cocaine doses. In contrast, ZFP189VPR treated mice do not experience changes in cocaine sensitivity and had lower rates of cocaine self-administration. Collectively, this research describes the brain mechanisms by which a TF specifically coordinates the molecular adaptations that produce increased cocaine addiction-like behaviors. The use of synthetic ZFP189VPR uncovers novel strategies for therapeutic interventions to potentially halt these cocaine-induced transcriptional processes.

Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity.

The p53 family of transcription factors (p53, p63 and p73) regulate diverse organismal processes including tumor suppression, maintenance of genome integrity and the development of skin and limbs. Crosstalk between transcription factors with highly similar DNA binding profiles, like those in the p53 family, can dramatically alter gene regulation. While p53 is primarily associated with transcriptional activation, p63 mediates both activation and repression. The specific mechanisms controlling p63-dependent gene regulatory activity are not well understood. Here, we use massively parallel reporter assays (MPRA) to investigate how local DNA sequence context influences p63-dependent transcriptional activity. Most regulatory elements with a p63 response element motif (p63RE) activate transcription, although binding of the p63 paralog, p53, drives a substantial proportion of that activity. p63RE sequence content and co-enrichment with other known activating and repressing transcription factors, including lineage-specific factors, correlates with differential p63RE-mediated activities. p63 isoforms dramatically alter transcriptional behavior, primarily shifting inactive regulatory elements towards high p63-dependent activity. Our analysis provides novel insight into how local sequence and cellular context influences p63-dependent behaviors and highlights the key, yet still understudied, role of transcription factor paralogs and isoforms in controlling gene regulatory element activity.

Expression interplay of genes coding for calcium-binding proteins and transcription factors during the osmotic phase provides insights on salt stress response mechanisms in bread wheat

Bread wheat is an important crop for the human diet, but the increasing soil salinization is reducing the yield. The Ca2+ signaling events at the early stages of the osmotic phase of salt stress are crucial for the acclimation response of the plants through the performance of calcium-sensing proteins, which activate or repress transcription factors (TFs) that affect the expression of downstream genes. Physiological, genetic mapping, and transcriptomics studies performed with the contrasting genotypes Syn86 (synthetic, salt-susceptible) and Zentos (elite cultivar, salt-tolerant) were integrated to gain a comprehensive understanding of the salt stress response. The MACE (Massive Analysis of cDNA 3ʹ-Ends) based transcriptome analysis until 4 h after stress exposure revealed among the salt-responsive genes, the over-representation of genes coding for calcium-binding proteins. The functional and structural diversity within this category was studied and linked with the expression levels during the osmotic phase in the contrasting genotypes. The non-EF-hand category from calcium-binding proteins was found to be enriched for the susceptibility response. On the other side, the tolerant genotype was characterized by a faster and higher up-regulation of genes coding for proteins with EF-hand domain, such as RBOHD orthologs, and TF members. This study suggests that the interplay of calcium-binding proteins, WRKY, and AP2/ERF TF families in signaling pathways at the start of the osmotic phase can affect the expression of downstream genes. The identification of SNPs in promoter sequences and 3ʹ -UTR regions provides insights into the molecular mechanisms controlling the differential expression of these genes through differential transcription factor binding affinity or altered mRNA stability.

Sialylated IgG induces the transcription factor REST in alveolar macrophages to protect against lung inflammation and severe influenza disease

While most respiratory viral infections resolve with little harm to the host, severe symptoms arise when infection triggers an aberrant inflammatory response that damages lung tissue. Host regulators of virally induced lung inflammation have not been well defined. Here, we show that enrichment for sialylated, but not asialylated immunoglobulin G (IgG), predicted mild influenza disease in humans and was broadly protective against heterologous influenza viruses in a murine challenge model. Mechanistic studies show that sialylated IgG mediated this protection by inducing the transcription factor repressor element-1 silencing transcription factor (REST), which repressed nuclear factor κB (NF-κB)-driven responses, preventing severe lung inflammation and protecting lung function during influenza infection. Therapeutic administration of a recombinant, sialylated Fc molecule in clinical development similarly activated REST and protected against severe influenza disease, demonstrating that this pathway could be clinically harnessed. Overall, induction of REST through sialylated IgG signaling is a strategy to limit inflammatory disease sequelae in infections caused by antigenically distinct influenza strains.

Multistability and predominant hybrid phenotypes in a four node mutually repressive network of Th1/Th2/Th17/Treg differentiation

Elucidating the emergent dynamics of cellular differentiation networks is crucial to understanding cell-fate decisions. Toggle switch – a network of mutually repressive lineage-specific transcription factors A and B – enables two phenotypes from a common progenitor: (high A, low B) and (low A, high B). However, the dynamics of networks enabling differentiation of more than two phenotypes from a progenitor cell has not been well-studied. Here, we investigate the dynamics of a four-node network A, B, C, and D inhibiting each other, forming a toggle tetrahedron. Our simulations show that this network is multistable and predominantly allows for the co-existence of six hybrid phenotypes where two of the nodes are expressed relatively high as compared to the remaining two, for instance (high A, high B, low C, low D). Finally, we apply our results to understand naïve CD4+ T cell differentiation into Th1, Th2, Th17 and Treg subsets, suggesting Th1/Th2/Th17/Treg decision-making to be a two-step process.

MicroRNAs provide negative feedback and stability in gene regulatory network models of cell-state transitions

The development of multicellular organisms occurs through a series of cell state transitions controlled by gene regulatory networks. Central to these networks are transcription factors (TFs) which bind enhancers and activate the expression of other genes, some of which are also TFs. Gene regulatory networks (GRN) connect TFs and enhancers in a nonlinear circuit capable of producing complex behavior such as bifurcations between stable cell states. Our dynamic network modelling of the Embryonic Stem Cell (ESC) to Definitive Endoderm (DE) transition requires an as yet unknown negative feedback mechanism for stability. Here, we show that cell state specific microRNAs (miRNAs) can provide this negative feedback by inactivating other cell lineage determining TFs (ESC or DE) during the transition. Our model provides a mechanism to maintain stable cell states without requiring a large set of cell-type-specific repressive TFs, of which there are fewer known examples than activators. In support of this model, we use computational models and analyze gene and miRNA expression and chromatin accessibility data from human cell lines to detect enhancers activating the miRNAs consistent with our network model. Our analysis highlights the interplay between TFs and miRNAs during ESC to DE transition and proposes a novel model for gene regulation.

Negative regulation of CcPAL2 gene expression by the repressor transcription factor CcMYB4-12 modulates lignin and capsaicin biosynthesis in Capsicum chinense fruits

Peppers globally renowned for their distinctive spicy flavor, have attracted significant research attention, particularly in understanding spiciness regulation. While the activator MYB's role in spiciness regulation is well-established, the involvement of repressor MYB factors remains unexplored. This study identified the MYB4 transcription factor through RNA-seq and genome-wide analysis as being associated with spiciness. Consequently, CcMYB4-2 and CcMYB4-12 were cloned from Hainan Huangdenglong peppers, both exhibiting nuclear subcellular localization. qRT-PCR analysis revealed that CcMYB4-2/4-12 had high expression levels during the accumulation period of capsaicin, but there were differences in their peak expression levels, which may be related to the formation of pepper spiciness. Heterologous expression in Arabidopsis thaliana resulted in significantly elevated CcMYB4-2/4-12 expression levels and reduced lignin content. In CcMYB4-2 silenced plants, PAL expression remained unchanged, while PAL expression significantly increased in CcMYB4-12 silenced plants, leading to elevated lignin content and reduced capsaicin content. Yeast one-hybrid (Y1H) and dual luciferase reporter assays (DLR) demonstrated that CcMYB4-2/4-12 inhibited the transcription of CcPAL2 by binding to its promoter. Notably, CcMYB4-12 exhibited more pronounced inhibition. Therefore, it is hypothesized that CcMYB4-12 plays a pivotal role in regulating lignin and capsaicin biosynthesis. This study elucidates the molecular mechanism of MYB4 binding to the PAL promoter, providing a foundational understanding for analyzing phenylpropanoid metabolism and its diverse branches. Key messageThrough functional verification analysis of the repressor CcMYB4, transcriptional regulation experiments revealed that CcMYB4 can bind to the CcPAL2 promoter, negatively regulating the capsaicin biosynthesis in Capsicum chinense fruits.

ZBTB46 coordinates angiogenesis and immunity to control tumor outcome.

Tumor angiogenesis and immunity show an inverse correlation in cancer progression and outcome1. Here, we report that ZBTB46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, controls both tumor angiogenesis and immunity. Zbtb46 was downregulated in both DCs and endothelial cells by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive conditions. Analysis of human cancer data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression led to TME changes to restrict tumor growth. Mechanistically, Zbtb46-deficient endothelial cells were highly angiogenic, and Zbtb46-deficient bone marrow progenitors upregulated Cebpb and diverted the DC program to immunosuppressive myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone marrow precursors toward immunostimulatory myeloid lineage output, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in preclinical models. These findings identify ZBTB46 as a critical factor for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

Genetic association and functional implications of TLR4 rs1927914 polymorphism on colon cancer risk

BackgroundColon cancer remains a major health concern worldwide, with genetic factors playing a crucial role in its development. Toll-like receptors (TLRs) has been implicated in various cancers, but their role in colon cancer is not well understood. This study aims to identify functional polymorphisms in the promoter and 3′UTR regions of TLRs and evaluate their association with colon cancer susceptibility.MethodsWe conducted a case-control study involving 410 colon cancer patients and 410 healthy controls from the Chinese population. Genotyping of polymorphisms in TLR3, TLR4, TLR5 and TLR7 was performed using PCR-RFLP and TaqMan MGB probes. Using logistic regression analysis, we evaluated the association of TLRs polymorphisms and the susceptibility to colon cancer. To understand the biological implications of the TLR4 rs1927914 polymorphism, we conducted functional assays, including luciferase reporter assay and electrophoretic mobility shift assay (EMSA).ResultsOur results demonstrated that the G-allele of the TLR4 rs1927914 polymorphism is significantly associated with a decreased risk of colon cancer (OR = 0.68, 95%CI = 0.50–0.91). Stratified analysis showed that TLR4 rs1927914 AG or GG genotype contributed to a decreased risk of colon cancer among younger individuals (OR = 0.52, 95%CI = 0.34–0.81), males (OR = 0.58, 95%CI = 0.38–0.87), non-smokers (OR = 0.58, 95%CI = 0.41–0.83) and non-drinker with OR (95%CI) of 0.66 (0.46–0.93). Functional assays demonstrated that in HCT116 and LOVO colon cancer cells, the luciferase activity driven by the TLR4 promoter with the rs1927914A allele was 5.43 and 2.07 times higher, respectively, compared to that driven by the promoter containing the rs1927914G allele. Electrophoretic mobility shift assay (EMSA) results indicated that the rs1927914G allele enhanced transcription factor binding. Using the transcription factor prediction tool, we found that the G allele facilitates binding of the repressive transcription factor Oct1, while the A allele does not.ConclusionThe TLR4 rs1927914 polymorphism influence the susceptibility to colon cancer, with the G allele offering a protective effect through modulation of gene expression. These insights enhance our understanding of the genetic determinants of colon cancer risk and highlight TLR4 as a promising target for cancer prevention strategies.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.