Gene-to-gene coordinated regulation of transcription and alternative splicing by 3D chromatin remodeling upon NF-κB activation.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

The NF-κB pathway is essential for coordinating gene expression in response to various stimuli, including viral infections. Most studies have focused on the role of NF-κB in transcriptional regulation. In the present study, the impact of the potent NF-κB activator HTLV-1 Tax oncoprotein on the three-dimensional organization of the genome was investigated. Tax-mediated NF-κB activation was found to restructure the 3D genomic landscape in cells and to bring genes together in multigene complexes that are coordinately regulated either transcriptionally or through alternative splicing by NF-κB. Induced coordinate changes in transcription and alternative splicing included the two master genes of NF-κB pathway NFKBIA and RELA. The findings have significant implications for understanding cell fate determination and disease development associated with HTLV-1 infection, as well as chronic NF-κB activation in various human inflammatory diseases and cancer.

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.

AMPK Associates with Chromatin and Phosphorylates the TAF-1 Subunit of the Transcription Initiation Complex to Regulate Histone Gene Expression in ALL Cells.

The survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL) remain poor. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK). To identify the target genes directly regulated by AMPKα2, we performed genome-wide RNA-seq and ChIP-seq in CCRF-CEM (T-ALL) cells expressing HA-AMPKα2 (CN2) under normal and energy/metabolic stress conditions. CN2 cells show significantly altered AMPKα2 genomic binding and transcriptomic profile under metabolic stress conditions, including reduced histone gene expression. Proteomic analysis and in vitro kinase assays identified the TATA-Box-Binding Protein-Associated Factor 1 (TAF1) as a novel AMPKα2 substrate that downregulates histone gene transcription in response to energy/metabolic stress. Knockdown and knockout studies demonstrated that both AMPKα2 and TAF1 are required for histone gene expression. Mechanistically, upon activation, AMPKα2 phosphorylates TAF1 at Ser-1353 which impairs TAF1 interaction with RNA polymerase II (Pol II), leading to a compromised state of p-AMPKα2/p-TAF1/Pol II chromatin association and suppression of transcription. This mechanism was also observed in primary ALL cells and in vivo in NSG mice. Consequently, we uncovered a non-canonical function of AMPK that phosphorylates TAF1, both members of a putative chromatin-associated transcription complex that regulate histone gene expression, among others, in response to energy/metabolic stress. IMPLICATIONS: Fully delineating the protein interactome by which AMPK regulates adaptive survival responses to energy/metabolic stress, either via epigenetic gene regulation or other mechanisms, will allow the rational development of strategies to overcome de novo or acquired resistance in ALL and other cancers.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

SIRT7 mediates L1 elements transcriptional repression and their association with the nuclear lamina.

Long interspersed elements-1 (LINE-1, L1) are retrotransposons that hold the capacity of self-propagation in the genome with potential mutagenic outcomes. How somatic cells restrict L1 activity and how this process becomes dysfunctional during aging and in cancer cells is poorly understood. L1s are enriched at lamin-associated domains, heterochromatic regions of the nuclear periphery. Whether this association is necessary for their repression has been elusive. Here we show that the sirtuin family member SIRT7 participates in the epigenetic transcriptional repression of L1 genome-wide in both mouse and human cells. SIRT7 depletion leads to increased L1 expression and retrotransposition. Mechanistically, we identify a novel interplay between SIRT7 and Lamin A/C in L1 repression. Our results demonstrate that SIRT7-mediated H3K18 deacetylation regulates L1 expression and promotes L1 association with elements of the nuclear lamina. The failure of such activity might contribute to the observed genome instability and compromised viability in SIRT7 knockout mice. Overall, our results reveal a novel function of SIRT7 on chromatin organization by mediating the anchoring of L1 to the nuclear envelope, and a new functional link of the nuclear lamina with transcriptional repression.

High pre-diagnosis attrition among patients with presumptive MDR-TB: an operational research from Bhopal district, India.

BACKGROUND: Pre-diagnosis attrition needs to be addressed urgently if we are to make progress in improving MDR-TB case detection and achieve universal access to MDR-TB care. We report the pre-diagnosis attrition, along with factors associated, and turnaround times related to the diagnostic pathway among patient with presumptive MDR-TB in Bhopal district, central India (2014). METHODS: Study was conducted under the Revised National Tuberculosis Control Programme setting. It was a retrospective cohort study involving record review of all registered TB cases in Bhopal district that met the presumptive MDR-TB criteria (eligible for DST) in 2014. In quarter 1, Line Probe Assay (LPA) was used if sample was smear/culture positive. Quarter 2 onwards, LPA and Cartridge-based Nucleic Acid Amplification Test (CbNAAT) was used for smear positive and smear negative samples respectively. Pre-diagnosis attrition was defined as failure to undergo DST among patients with presumptive MDR-TB (as defined by the programme). RESULTS: Of 770 patients eligible for DST, 311 underwent DST and 20 patients were diagnosed as having MDR-TB. Pre-diagnosis attrition was 60% (459/770). Among those with pre-diagnosis attrition, 91% (417/459) were not identified as 'presumptive MDR-TB' by the programme. TAT [median (IQR)] to undergo DST after eligibility was 4 (0, 10) days. Attrition was more than 40% across all subgroups. Age more than 64 years; those from a medical college; those eligible in quarter 1; patients with presumptive criteria 'previously treated - recurrent TB', 'treatment after loss-to-follow-up' and 'previously treated-others'; and patients with extra-pulmonary TB were independent risk factors for not undergoing DST. CONCLUSION: High pre-diagnosis attrition was contributed by failure to identify and refer patients. Attrition reduced modestly with time and one factor that might have contributed to this was introduction of CbNAAT in quarter 2 of 2014. General health system strengthening which includes improvement in identification/referral and patient tracking with focus on those with higher risk for not undergoing DST is urgently required.

Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells.

While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks.

Chromatin profiling reveals regulatory network shifts and a protective role for hepatocyte nuclear factor 4α during colitis.

Transcriptional regulatory mechanisms likely contribute to the etiology of inflammatory bowel disease (IBD), as genetic variants associated with the disease are disproportionately found at regulatory elements. However, the transcription factors regulating colonic inflammation are unclear. To identify these transcription factors, we mapped epigenomic changes in the colonic epithelium upon inflammation. Epigenetic marks at transcriptional regulatory elements responded dynamically to inflammation and indicated a shift in epithelial transcriptional factor networks. Active enhancer chromatin structure at regulatory regions bound by the transcription factor hepatocyte nuclear factor 4α (HNF4A) was reduced during colitis. In agreement, upon an inflammatory stimulus, HNF4A was downregulated and showed a reduced ability to bind chromatin. Genetic variants that confer a predisposition to IBD map to HNF4A binding sites in the human colon cell line CaCo2, suggesting impaired HNF4A binding could underlie genetic susceptibility to IBD. Despite reduced HNF4A binding during inflammation, a temporal knockout model revealed HNF4A still actively protects against inflammatory phenotypes and promotes immune regulatory gene expression in the inflamed colonic epithelium. These findings highlight the potential for HNF4A agonists as IBD therapeutics.

Changing the DNA recognition specificity of the EcoDam DNA-(adenine-N6)-methyltransferase by directed evolution.

EcoDam is an adenine-N6 DNA methyltransferase that methylates the GATC sites in the Escherichia coli genome. We have changed the target specificity of EcoDam from GATC to GATT by directed evolution, combining different random mutagenesis methods with restriction protection at GATT sites for selection and screening. By co-evolution of an enzyme library and a substrate library, we identified GATT as the best non-GATC site and discover a double mutation, R124S/P134S, as the first step to increase enzyme activity at GATT sites. After four generations of mutagenesis and selection, we obtained enzyme variants with new specificity for GATT. While the wild-type EcoDam shows no detectable activity at GATT sites in E. coli cells, some variants prefer methylation at GATT over GATC sites by about 10-fold in cells. In vitro DNA methylation kinetics carried out under single-turnover conditions using a hemimethylated GATC and a GATT oligonucleotide substrate confirmed that the evolved proteins prefer methylation of GATT sites to a similar degree. They show up to 1600-fold change in specificity in vitro and methylate the new GATT target site with 20% of the rate of GATC methylation by the wild-type enzyme, indicating good activity. We conclude that the new methyltransferases are fully functional in vivo and in vitro but show a new target-site specificity.

Transition from EcoDam to T4Dam DNA recognition mechanism without loss of activity and specificity.

The EcoDam and T4Dam DNA-(adenine N6)-methyltransferases both methylate the adenine residue in GATC sites. These enzymes are highly related in amino acid sequence, but they deviate in their contact to the first base pair of the target sequence. EcoDam contacts Gua1 with K9 (which corresponds to T4Dam A6), while T4Dam contacts Gua1 with R130 (which corresponds to EcoDam Y138). We have "transplanted" the T4Dam DNA recognition into EcoDam and show that the EcoDam K9A/Y138R double mutant is highly active and specific. We also studied the intermediates of this transition: The EcoDam K9A variant showed low activity and loss of recognition of Gua1 [Horton, et al., J. Mol. Biol. 2006, 358, 559-570]. In contrast, the EcoDam Y138R variant, which carries both Gua1 recognition elements (K9 from EcoDam and R138 corresponding to R130 from T4Dam), is fully active and specific. This result indicates that a smooth evolutionary pathway exists for changing the EcoDam DNA recognition mode to T4Dam without loss of activity and without generation of evolutionary intermediates with reduced activity. We consistently observed increased activity of EcoDam variants containing Y138R; this suggests that the transition from EcoDam (Gua1 recognition through K9) to T4Dam (Gua1 recognition through R130) was driven by selective pressure towards increased catalytic activity.

Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle.

The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)).