Mechanistic and kinetic insights into Transcription factor biology via acute protein depletion.

Genetic downregulation of the BCL11A transcription factor reverses the switch from fetal to adult hemoglobin and is effective in treating ß-hemoglobinopathies. Genetic ablation results in gradual reduction in protein abundance and does not lend itself to analysis of immediate consequences of protein loss or determination of the direct interactors/targets of the protein of interest. We achieved acute degradation of the largely disordered and 'undruggable' BCL11A protein by fusing it with a conditional degradation (degron) tag, FKBP12F36V, called dTAG. Small molecules then depleted the BCL11A-dTAG through endogenous proteolytic pathways. By integrating acute depletion with nascent transcriptomics and cell cycle separation techniques, we demonstrate the necessity of BCL11A occupancy at target chromatin for sustained transcriptional repression in erythroid cells. We advocate for expanding the exploration of transcription factor function to include acute depletion, which holds the potential to unveil unprecedented kinetic insights into TF mechanisms of action.

Matrin3 mediates differentiation through stabilizing chromatin loop-domain interactions and YY1 mediated enhancer-promoter interactions.

Although emerging evidence indicates that alterations in proteins within nuclear compartments elicit changes in chromosomal architecture and differentiation, the underlying mechanisms are not well understood. Here we investigate the direct role of the abundant nuclear complex protein Matrin3 (Matr3) in chromatin architecture and development in the context of myogenesis. Using an acute targeted protein degradation platform (dTAG-Matr3), we reveal the dynamics of development-related chromatin reorganization. High-throughput chromosome conformation capture (Hi-C) experiments revealed substantial chromatin loop rearrangements soon after Matr3 depletion. Notably, YY1 binding was detected, accompanied by the emergence of novel YY1-mediated enhancer-promoter loops, which occurred concurrently with changes in histone modifications and chromatin-level binding patterns. Changes in chromatin occupancy by Matr3 also correlated with these alterations. Overall, our results suggest that Matr3 mediates differentiation through stabilizing chromatin accessibility and chromatin loop-domain interactions, and highlight a conserved and direct role for Matr3 in maintenance of chromosomal architecture.

Leukemia core transcriptional circuitry is a sparsely interconnected hierarchy stabilized by incoherent feed-forward loops.

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators' direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.

High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines.

The dTAG system is a versatile strategy for tunable control of protein abundance and facilitates the time-resolved assessment of disease-associated protein function. A "co-opted" fusion-based degron peptide, the "dTAG" facilitates the study of endogenous protein function when knocked-in at the endogenous genetic loci of proteins of interest. We combine CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with the delivery of a single-stranded DNA HDR-donor-template via crude preparations of recombinant adeno-associated virus (rAAV). Our approach to knock-in of large (1-2kb) DNA fragments via crude-rAAV mediated HDR donor delivery is rapid and inexpensive. It facilitates genetic modification of a variety of human as well as mouse cell lines at high efficiency and precision.

A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes Treg cell activation and homeostasis.

The molecular programs involved in regulatory T (Treg) cell activation and homeostasis remain incompletely understood. Here, we show that T cell receptor (TCR) signaling in Treg cells induces the nuclear translocation of serine/threonine kinase 4 (Stk4), leading to the formation of an Stk4-NF-κB p65-Foxp3 complex that regulates Foxp3- and p65-dependent transcriptional programs. This complex was stabilized by Stk4-dependent phosphorylation of Foxp3 on serine-418. Stk4 deficiency in Treg cells, either alone or in combination with its homolog Stk3, precipitated a fatal autoimmune lymphoproliferative disease in mice characterized by decreased Treg cell p65 expression and nuclear translocation, impaired NF-κB p65-Foxp3 complex formation, and defective Treg cell activation. In an adoptive immunotherapy model, overexpression of p65 or the phosphomimetic Foxp3S418E in Stk3/4-deficient Treg cells ameliorated their immune regulatory defects. Our studies identify Stk4 as an essential TCR-responsive regulator of p65-Foxp3-dependent transcription that promotes Treg cell-mediated immune tolerance.

Temporal resolution of gene derepression and proteome changes upon PROTAC-mediated degradation of BCL11A protein in erythroid cells.

Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for ß-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.

Maintenance of macrophage transcriptional programs and intestinal homeostasis by epigenetic reader SP140.

Epigenetic "readers" that recognize defined posttranslational modifications on histones have become desirable therapeutic targets for cancer and inflammation. SP140 is one such bromodomain- and plant homeodomain (PHD)-containing reader with immune-restricted expression, and single-nucleotide polymorphisms (SNPs) within SP140 associate with Crohn's disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unclear. We show that SP140 is critical for transcriptional programs that uphold the macrophage state. SP140 preferentially occupies promoters of silenced, lineage-inappropriate genes bearing the histone modification H3K27me3, such as the HOXA cluster in human macrophages, and ensures their repression. Depletion of SP140 in mouse or human macrophages resulted in severely compromised microbe-induced activation. We reveal that peripheral blood mononuclear cells (PBMCs) or B cells from individuals carrying CD-associated SNPs within SP140 have defective SP140 messenger RNA splicing and diminished SP140 protein levels. Moreover, CD patients carrying SP140 SNPs displayed suppressed innate immune gene signatures in a mixed population of PBMCs that stratified them from other CD patients. Hematopoietic-specific knockdown of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)-induced colitis, and low SP140 levels in human CD intestinal biopsies correlated with relatively lower intestinal innate cytokine levels and improved response to anti-tumor necrosis factor (TNF) therapy. Thus, the epigenetic reader SP140 is a key regulator of macrophage transcriptional programs for cellular state, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by ineffective innate immunity, normally critical for intestinal homeostasis.

Transcription driven somatic DNA methylation within the imprinted Gnas cluster.

Differential marking of genes in female and male gametes by DNA methylation is essential to genomic imprinting. In female gametes transcription traversing differentially methylated regions (DMRs) is a common requirement for de novo methylation at DMRs. At the imprinted Gnas cluster oocyte specific transcription of a protein-coding transcript, Nesp, is needed for methylation of two DMRs intragenic to Nesp, namely the Nespas-Gnasxl DMR and the Exon1A DMR, thereby enabling expression of the Gnas transcript and repression of the Gnasxl transcript. On the paternal allele, Nesp is repressed, the germline DMRs are unmethylated, Gnas is repressed and Gnasxl is expressed. Using mutant mouse models, we show that on the paternal allele, ectopic transcription of Nesp traversing the intragenic Exon1A DMR (which regulates Gnas expression) results in de novo methylation of the Exon1A DMR and de-repression of Gnas just as on the maternal allele. However, unlike the maternal allele, methylation on the mutant paternal allele occurs post-fertilisation, i.e. in somatic cells. This, to our knowledge is the first example of transcript/transcription driven DNA methylation of an intragenic CpG island, in somatic tissues, suggesting that transcription driven de novo methylation is not restricted to the germline in the mouse. Additionally, Gnasxl is repressed on a paternal chromosome on which Nesp is ectopically expressed. Thus, a paternally inherited Gnas cluster showing ectopic expression of Nesp is "maternalised" in terms of Gnasxl and Gnas expression. We show that these mice have a phenotype similar to mutants with two expressed doses of Gnas and none of Gnasxl.

Beyond receptors and signaling: epigenetic factors in the regulation of innate immunity.

The interaction of innate immune cells with pathogens leads to changes in gene expression that elicit our body's first line of defense against infection. Although signaling pathways and transcription factors have a central role, it is becoming increasingly clear that epigenetic factors, in the form of DNA or histone modifications, as well as noncoding RNAs, are critical for generating the necessary cell lineage as well as context-specific gene expression in diverse innate immune cell types. Much of the epigenetic landscape is set during cellular differentiation; however, pathogens and other environmental triggers also induce changes in histone modifications that can either promote tolerance or 'train' innate immune cells for a more robust antigen-independent secondary response. Here we review the important contribution of epigenetic factors to the initiation, maintenance and training of innate immune responses. In addition, we explore how pathogens have hijacked these mechanisms for their benefit and the potential of small molecules targeting chromatin machinery as a way to boost or subdue the innate immune response in disease.

Antisense Activity across the Nesp Promoter is Required for Nespas-Mediated Silencing in the Imprinted Gnas Cluster.

Macro long non-coding RNAs (lncRNAs) play major roles in gene silencing in inprinted gene clusters. Within the imprinted Gnas cluster, the paternally expressed Nespas lncRNA downregulates its sense counterpart Nesp. To explore the mechanism of action of Nespas, we generated two new knock-in alleles to truncate Nespas upstream and downstream of the Nesp promoter. We show that Nespas is essential for methylation of the Nesp differentially methylated region (DMR), but higher levels of Nespas are required for methylation than are needed for downregulation of Nesp. Although Nespas is transcribed for over 27 kb, only Nespas transcript/transcription across a 2.6 kb region that includes the Nesp promoter is necessary for methylation of the Nesp DMR. In both mutants, the levels of Nespas were extraordinarily high, due at least in part to increased stability, an effect not seen with other imprinted lncRNAs. However, even when levels were greatly raised, Nespas remained exclusively cis-acting. We propose Nespas regulates Nesp methylation and expression to ensure appropriate levels of expression of the protein coding transcripts Gnasxl and Gnas on the paternal chromosome. Thus, Nespas mediates paternal gene expression over the entire Gnas cluster via a single gene, Nesp.

Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.

MTHFR C677T and A1298C polymorphisms are risk factors for Down's syndrome in Indian mothers.

Down's syndrome (DS), a chromosomal disorder due to trisomy 21, results mostly from nondisjunction in maternal meiosis. The present case-control study examined the association of genetic polymorphisms with predisposition to nondisjunction. Two common polymorphisms (SNPs), C677T and A1298C, in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene involved in folate metabolism, are known to lower the activity of this enzyme. Three hundred and fourteen mothers (with DS children and controls), mostly from the eastern states of India, were genotyped for the two above-mentioned SNPs. Significant association with both of these SNPs were detected, more specifically, in the mothers of DS children homozygous for the polymorphic alleles 677 T and 1298 C. The relative risk of T (C677T) and C (A1298C) homozygosity in mothers for DS-affected pregnancy was 7 (OR 7.67, 95% CI 1.67-35.08, P=0.003) and 4 (OR 4.40, 95% CI 1.45-13.26, P=0.008), respectively. Moreover, all 677TT mothers studied were less than 31 years of age, whereas no correlation with maternal age was observed for A1298C genotypes. Interestingly, all of the young 677TT mothers had either a first- or secondborn child with DS. Thus, this study reports that young Indian mothers with TT genotypes are genetically predisposed to nondisjunction due to abnormal folate metabolism.