Sanjeev Shukla

CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

Co-transcriptional regulation of alternative pre-mRNA splicing

While studies of alternative pre-mRNA splicing regulation have typically focused on RNA-binding proteins and their target sequences within nascent message, it is becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II) elongation and chromatin structure are intricately intertwined. The majority of introns in higher eukaryotes are excised prior to transcript release in a manner that is dependent on transcription through pol II. As a result of co-transcriptional splicing, variations in pol II elongation influence alternative splicing patterns, wherein a slower elongation rate is associated with increased inclusion of alternative exons within mature mRNA. Physiological barriers to pol II elongation, such as repressive chromatin structure, can thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can reciprocally influence pol II elongation and chromatin structure. Here, we highlight recent advances in co-transcriptional splicing that reveal an extensive network of coupling between splicing, transcription and chromatin remodeling complexes. This article is part of a Special Issue entitled: Chromatin in time and space.

TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4+ T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins.

Immunoproteomics reveals that cancer of the tongue and the gingivobuccal complex exhibit differential autoantibody response.

Autoantibody response to tumor antigens has been widely used to identify novel tumor markers for different cancers, including that of the head and neck. The oral cavity, which is in the head and neck region, comprises of many sub sites with distinct biologies and incidence of cancer of each sub site of the oral cavity is different. It is anticipated therefore that each sub site of the oral cavity may elicit a differential autoantibody response. This report evaluates the autoantibody response in 15 patients with cancer of gingivo-buccal complex and in 15 patients with cancer of tongue using Immunoproteomics, and shows that the autoantibody response to alpha-enolase, HSP 70, peroxiredoxin-VI, annexin II, pyruvate kinase, alpha-tubulin, beta-tubulin, ATP synthase, triose phosphate isomerase and aldose reductase seen in patients with cancer of gingivo-buccal complex is absent in patients with cancer of tongue. This suggests that cancer of these sub sites should be studied separately because of their different biology and emerging site specific molecular signatures including autoantibody responses to ensure unambiguous clinical interpretations.

Tumor antigens eliciting autoantibody response in cancer of gingivo-buccal complex.

Cancer of the gingivo‐buccal complex (GBC) is a major cancer in Indian men. This study reports the identification of tumor antigens, which elicit an antibody response in cancer of GBC using immunoproteomics. Proteins from KB cells separated by 2‐D PAGE, were immunoblotted with IgG from sera of 28 cancer patients, 12 patients with leukoplakia, and 28 healthy individuals. Antigens detected by the IgGs from the patients sera were different among different individuals with presence of any single antigen ranging from 7 to 79%. Several of these antigens have been identified by MS and confirmed by immunostaining. They are three forms of α‐enolase, peroxiredoxin‐VI, annexin‐II, HSP70, pyruvate kinase, α‐tubulin, β‐tubulin, ATP‐synthase, phosphoglycerate mutase (PGM), aldose reductase, triosephosphate isomerase, and cyclophilin‐A. Except, HSP70, these antigens are being reported in cancer of GBC for the first time. Pyruvate kinase and aldose reductase have not been reported to elicit autoantibody response in any other cancer earlier. Initial results show that autoantibody response against α‐enolase, HSP70, annexin‐II, peroxiredoxin‐VI, and aldose reductase are also seen in patients with leukoplakia of GBC, which suggest early occurrence of these autoantibodies during the process of oral carcinogenesis. These antigens can be further validated for their use in cancer management by immune intervention.

Integrated genomic analyses identify KDM1A's role in cell proliferation via modulating E2F signaling activity and associate with poor clinical outcome in oral cancer.

The histone demethylase KDM1A specifically demethylates lysine residues and its deregulation has been implicated in the initiation and progression of various cancers. However, KDM1As molecular role and its pathological consequences, and prognostic significance in oral cancer remain less understood. In the present study, we sought to investigate the expression of KDM1A and its downstream role in oral cancer pathogenesis. By comparing mRNA expression profiles, we identified an elevated KDM1A expression in oral tumors when compared to normal oral tissues. In silico pathway prediction identified the association between KDM1A and E2F1 signaling in oral cancer. Pathway scanning, functional annotation analysis and In vitro assays showed the KDM1As involvement in oral cancer cell proliferation and the cell cycle. Moreover, real time PCR and luciferase assays confirmed KDM1As role in regulation of E2F1 signaling activity in oral cancer. Elevated KDM1A expression is associated with poor clinical outcome in oral cancer. Our data indicate that deregulated KDM1A expression is positively associated with proliferative phenotype of oral cancer and confers poor clinical outcome. These cumulative data suggest that KDM1A might be a potential diagnostic and therapeutic target for oral cancer.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

Significance Recent advances in cancer epigenetics have shown the involvement of epigenetic abnormalities in the initiation and progression of cancer, but their role in cancer-specific aberrant splicing is not clear. The identification of upstream epigenetic regulators of cancer-specific splicing will enable us to therapeutically target aberrant splicing and provide an approach to cancer therapy. Here we have demonstrated a mechanism of intragenic DNA methylation-mediated regulation of alternative splicing by Brother of Regulator of Imprinted Sites (BORIS), which can contribute to breast cancer tumorigenesis by favoring the Warburg effect. The reversal of the Warburg effect was achieved by the inhibition of DNA methylation or down-regulation of BORIS, which may serve as a useful approach to inhibit the growth of breast cancer cells. Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.

Prognostic utility of autoantibodies to α-enolase and Hsp70 for cancer of the gingivo-buccal complex using immunoproteomics

Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC.

PAK2–c-Myc–PKM2 axis plays an essential role in head and neck oncogenesis via regulating Warburg effect

The histone modifiers (HMs) are crucial for chromatin dynamics and gene expression; however, their dysregulated expression has been observed in various abnormalities including cancer. In this study, we have analyzed the expression of HMs in microarray profiles of head and neck cancer (HNC), wherein a highly significant overexpression of p21-activated kinase 2 (PAK2) was identified which was further validated in HNC patients. The elevated expression of PAK2 positively correlated with enhanced cell proliferation, aerobic glycolysis and chemoresistance and was associated with the poor clinical outcome of HNC patients. Further, dissection of molecular mechanism revealed an association of PAK2 with c-Myc and c-Myc-dependent PKM2 overexpression, wherein we showed that PAK2 upregulates c-Myc expression and c-Myc thereby binds to PKM promoter and induces PKM2 expression. We observed that PAK2–c-Myc–PKM2 axis is critical for oncogenic cellular proliferation. Depletion of PAK2 disturbs the axis and leads to downregulation of c-Myc and thereby PKM2 expression, which resulted in reduced aerobic glycolysis, proliferation and chemotherapeutic resistance of HNC cells. Moreover, the c-Myc complementation rescued PAK2 depletion effects and restored aerobic glycolysis, proliferation, migration and invasion in PAK2-depleted cells. The global transcriptome analysis of PAK2-depleted HNC cells revealed the downregulation of various genes involved in active cell proliferation, which indicates that PAK2 overexpression is critical for HNC progression. Together, these results suggest that the axis of PAK2–c-Myc–PKM2 is critical for HNC progression and could be a therapeutic target to reduce the cell proliferation and acquired chemoresistance and might enhance the efficacy of standard chemotherapy which will help in better management of HNC patients.