Biology subjects

Nizamuddin, S.

Publications and source records attributed to Nizamuddin, S..

7 recordsLinked to original sources

bioRxiv2026

SMAD4 MH2 Mutations Disrupt CREBBP/EP300 Recruitment and TGF-β-Induced Transcription in Colorectal Cancer

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@15d4cborg.highwire.dtl.DTLVardef@14591d6org.highwire.dtl.DTLVardef@69990dorg.highwire.dtl.DTLVardef@bf5e23_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing

TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

molecular biology↗

bioRxiv2023

Alternative mRNA splicing controls the functions of the histone H3K27 demethylase UTX/KDM6A

BackgroundThe UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. MethodsUsing Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer and normal epithelium. ResultsThe central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8-48% in bladder tissues and 18-58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, hence localize to the nucleus, unlike cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon 14-containing isoform compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. ConclusionUTX mRNAs undergo extensive alternative splicing that controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes. Simple SummaryUTX/KDM6A is a histone H3K27 demethylase and plays an important role in mammalian development and human diseases such as urothelial cancer. We identified a region encompassing exons 12-17 of UTX that undergoes extensive splicing events. As a result, a nuclear localization sequence located in exon14 is missing in a considerable part of UTX transcripts in different cell lines and tissues from normal bladder epithelium and bladder cancer. Mass spectrometry analysis showed a role for this region in binding to the epigenetic PR-DUB and MiDAC complexes. UTX was also more extensively bound to chromatin when the alternative splicing region presented. Our study showed that alternative splicing of UTX transcripts plays an important role in its functions.

biochemistry↗

bioRxiv2022

Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

Glioblastoma is the most common and aggressive primary brain tumor with poor prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma.

cancer biology↗

bioRxiv2022

Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity

Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. After in silico screening of 253 disease-related MEN1 missense mutations, we selected a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the interaction with MLL1 compared to JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. These findings support the general importance of the menin-MLL1 and menin-JunD interactions in MEN1 gene-associated pathogenic conditions.

molecular biology↗

bioRxiv2017

Genetic polymorphism of Cytochrome-P450-2C9 (CYP2C9) in Indian populations

Cytochrome-P450-2C9 (CYP2C9) metabolizes wide range of drugs and highly express in human liver. Various mutations of CYP2C9 (R144C, I359L etc.), associated with drug-response, are highly diverse. We aimed to investigate the genetic diversity of CYP2C9 in Indian-subcontinent, using 1278 subjects from 36 populations. High frequency of CYP2C9*3 (0-0.179) was observed, comparative to other populations, including Europeans. Subjects having CYP2C9*3/*3 requires lower dose of warfarin, comparative to CYP2C9*1/*3 or CYP2C9*1/*1. Since, Indians are practicing marriage among their caste system, we predicted and observed high frequency (0-0.05) of CYP2C9*3/*3. Out of 21 populations, living outside of Indian subcontinent, only Toscani and Southern Han-Chinese have 0.009 and 0.01 CYP2C9*3/*3, respectively, lower than Indians. We found a non-synonymous mutation (L362V), observed only in Indian-subcontinent, and have 0-0.056 allelic, 0-0.037 L/V and 00.037 V/V genotype frequency. We observed unfavorable interatomic interactions between hydroxylation sites of warfarin and reactive oxyferryl heme in mutant, comparative to wild-type CYP2C9, in molecular dynamic simulations; and predict lower kinetic activity.

evolutionary biology↗

bioRxiv2017

Signatures of natural selection in the drug metabolizing enzyme genes: Opportunity for developing personalized and precision medicine

Modern human experienced various selective pressures; including range of xenobiotics which contributed to heterogeneity of drug response. Many genes involve in pharmacokinetics and dynamics of drug, have been reported under natural selection. However, none of the studies have utilized comprehensive information of drug-centered PharmGKB pathways. We have extended this work and aimed to investigate sweep signals, using 1,798 subjects, from 53 Indian and 15 other world populations. We observed that modifiers which alters the biochemical function of other genes, have excess of natural selection (median std-z score=0.033{+/-}0.95; p-value=1.7x10-9-3.7x10-3). Taxane and statin primarily used for chemotherapy and lowering cholesterol level, respectively; and well known for heterogeneous drug response. We observed that pharmacokinetic pathway of taxane and statins are under natural selection (p-value=2.53x10-9and 2.73x10-9-1.09x10-4; q-value=1.28x10-7 and 6.91x10-6-1.1x10-3). We also observed signal of selection in Ibuprofen pharmacokinetics (p-value=1.76x10-5; q-value =2.22x10-4), beta-agonist/beta-blocker pharmacodynamics (p-value=4.79x10-4; q-value =4.04x10-4) and Zidovudin pharmacokinetics/dynamic pathway (p-value=7.0x10-4; q-value =5.06x10-4). Hard sweeps signals were observed in a total of 322 loci. Of which, 53 affect mRNA expression (p-value<0.001) and 16 were already reported with therapeutic response. Interestingly, we observed that Africans have experience 2 phases of natural selection, one at ~30,000 another at ~10,000 years before present.

evolutionary biology↗