Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

NCoR1 controls Mycobacterium tuberculosis growth in myeloid cells by regulating the AMPK-mTOR-TFEB axis.

Mycobacterium tuberculosis (Mtb) defends host-mediated killing by repressing the autophagolysosome machinery. For the first time, we report NCoR1 co-repressor as a crucial host factor, controlling Mtb growth in myeloid cells by regulating both autophagosome maturation and lysosome biogenesis. We found that the dynamic expression of NCoR1 is compromised in human peripheral blood mononuclear cells (PBMCs) during active Mtb infection, which is rescued upon prolonged anti-mycobacterial therapy. In addition, a loss of function in myeloid-specific NCoR1 considerably exacerbates the growth of M. tuberculosis in vitro in THP1 differentiated macrophages, ex vivo in bone marrow-derived macrophages (BMDMs), and in vivo in NCoR1MyeKO mice. We showed that NCoR1 depletion controls the AMPK-mTOR-TFEB signalling axis by fine-tuning cellular adenosine triphosphate (ATP) homeostasis, which in turn changes the expression of proteins involved in autophagy and lysosomal biogenesis. Moreover, we also showed that the treatment of NCoR1 depleted cells by Rapamycin, Antimycin-A, or Metformin rescued the TFEB activity and LC3 levels, resulting in enhanced Mtb clearance. Similarly, expressing NCoR1 exogenously rescued the AMPK-mTOR-TFEB signalling axis and Mtb killing. Overall, our data revealed a central role of NCoR1 in Mtb pathogenesis in myeloid cells.

A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

CMTM6 attenuates cisplatin-induced cell death in OSCC by regulating AKT/c-Myc-driven ribosome biogenesis.

CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism of how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin-resistant OSCC lines stably expressing Nt shRNA and CMTM6 shRNA revealed that CMTM6 might be a potential regulator of the ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over-expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 was also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin-resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or pharmacological inhibitor (CX-5461) significantly restores cisplatin-mediated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of the ribosome biogenesis network and targeting the ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.

Epigenomics of conventional type-I dendritic cells depicted preferential control of TLR9 versus TLR3 response by NCoR1 through differential IRF3 activation.

Tight control of gene regulation in dendritic cells (DCs) is important to mount pathogen specific immune responses. Apart from transcription factor binding, dynamic regulation of enhancer activity through global transcriptional repressors like Nuclear Receptor Co-repressor 1 (NCoR1) plays a major role in fine-tuning of DC responses. However, how NCoR1 regulates enhancer activity and gene expression in individual or multiple Toll-like receptor (TLR) activation in DCs is largely unknown. In this study, we did a comprehensive epigenomic analysis of murine conventional type-I DCs (cDC1) across different TLR ligation conditions. We profiled gene expression changes along with H3K27ac active enhancers and NCoR1 binding in the TLR9, TLR3 and combined TLR9 + TLR3 activated cDC1. We observed spatio-temporal activity of TLR9 and TLR3 specific enhancers regulating signal specific target genes. Interestingly, we found that NCoR1 differentially controls the TLR9 and TLR3-specific responses. NCoR1 depletion specifically enhanced TLR9 responses as evident from increased enhancer activity as well as TLR9-specific gene expression, whereas TLR3-mediated antiviral response genes were negatively regulated. We validated that NCoR1 KD cDC1 showed significantly decreased TLR3 specific antiviral responses through decreased IRF3 activation. In addition, decreased IRF3 binding was observed at selected ISGs leading to their decreased expression upon NCoR1 depletion. Consequently, the NCoR1 depleted cDC1 showed reduced Sendai Virus (SeV) clearance and cytotoxic potential of CD8+ T cells upon TLR3 activation. NCoR1 directly controls the majority of these TLR specific enhancer activity and the gene expression. Overall, for the first time, we revealed NCoR1 mediates transcriptional control towards TLR9 as compared to TLR3 in cDC1.

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy.

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress-induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62-NRF2 axis. We show that TRIM16 is an integral part of the p62-KEAP1-NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress-induced aggregate clearance and protects cells against oxidative/proteotoxic stress-induced toxicity in vitro and in vivo Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation-associated diseases such as neurodegeneration.

Zbtb10 transcription factor is crucial for murine cDC1 activation and cytokine secretion.

Dendritic cell (DC) activation and cytokine production is tightly regulated. In this study, we found that Zbtb10 expression is activation dependent and it is essential for the immunogenic function of cDC1. Zbtb10 knockdown (KD) significantly reduced the expression of co-stimulatory genes CD80 and CD86 along with cytokines including IL-12, IL-6, and IL-10, in activated cDC1 Mutu-DC line. Consequently, the clonal expansion of CD44+ effector T cells in co-cultured CD4+ T cells was drastically reduced owing to significantly reduced IL-2. At the same time, these CD44+ effector T cells were unable to differentiate toward Tbet+ IFNγ+ Th1 subtype. Instead, an increased frequency of Th2 cells expressing GATA3+ and IL-13+ was observed. Interestingly, in Zbtb10 KD condition the co-cultured T cells depicted increased expression of PD1 and LAG3, the T-cell anergic markers. Moreover, the global transcriptome analysis identified that Zbtb10 is pertinent for DC activation and its depletion in cDC1 completely shuts down their immune responses. Mechanistic analysis revealed that Zbtb10 KD enhanced the expression of NKRF (NF-κB repressing factor) leading to drastic suppression of NF-κB related genes. Zbtb10 KD abrogated p65 and RelB nuclear translocation, thereby controlling the activation and maturation of cDC1 and the ensuing adaptive T cell responses.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

Toll-like receptor-7 activation in CD8+ T cells modulates inflammatory mediators in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder of unknown etiology with aberrant immunological responses leading to inflammation, swelling and pain of the joints. CD8+ T cells have been known to be one of the major immune modulators in the progression of RA and the presence of toll-like receptors (TLRs) on these cells further accentuate their role in RA. Herein, we report an increased expression of TLR7 in the endosomes of CD8+ T cells of RA patients correlating with disease severity. The stimulation of TLR7 with Imiquimod (IMQ) in these CD8+ T cells drives the signalling cascade via NFkB and pERK activation and hence an increase in the mRNA transcripts of signature cytokines and cytolytic enzymes. However, a parallel synthesis of Tristetraprolin (TTP), an mRNA destabilizing protein prevents the translation of the mRNA transcripts, leading to a rapid degeneration of the target mRNA. We thus report that a direct TLR7 ligation by its agonist increases cytokine transcript signature but not an equivalent protein surge.

NCoR1 fine-tunes type-I IFN response in cDC1 dendritic cells by directly regulating Myd88-IRF7 axis under TLR9.

Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-ß expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-ß-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-ß secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC-/- animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.

Transcriptional regulatory logic of the diurnal cycle in the mouse liver.

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.

Integrative genomics identifies the corepressor SMRT as a gatekeeper of adipogenesis through the transcription factors C/EBPß and KAISO.

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPß-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic ß-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and ßTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in ßTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate ß-cell function and may play a critical role in the development of diabetes.

Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages.

Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette-Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.