Biology subjects

Gdula, M. R.

Publications and source records attributed to Gdula, M. R..

4 recordsLinked to original sources

bioRxiv2025

Proximity of pre-mRNA 3' end processing and transcription termination predicts enhanced gene expression

Two key factors required for pre-mRNA 3 end cleavage and polyadenylation, CPSF73 and PCF11, exhibit oncogenic properties: their elevated expression is associated with poor cancer patient prognosis. However, both proteins are also transcription termination factors, and it is unclear which of the processes they promote might contribute to carcinogenesis. Here, we employ a cellular model of colorectal cancer (CRC) progression and find that cells from primary tumor are addicted to high levels of CPSF73 and PCF11, while metastatic cells become less sensitive to their levels. We find no association between alternative polyadenylation (APA) and cell dependence on CPSF73 and PCF11, and no impact of their downregulation on transcription-replication collisions. Instead, we uncover an uncoupling of changes in 3 cleavage and termination during CRC progression: primary tumor-derived cells display a global shift to more proximal termination, yet a tendency for distal APA, compared to normal cells. Metastatic cells display partial reversion toward termination patterns observed in normal cells, and opposite tendency favoring proximal APA. This prompts us to measure the distance between the site of 3 cleavage and transcription termination for active protein-coding genes and find it almost halved in cells from primary tumor compared to normal cells. Interestingly, this distance becomes critically short for oncogenes. Closer proximity of 3 cleavage to termination correlates with higher gene expression, both across genes within a cell line and when distance and expression change in parallel. This uncovers a new relationship of transcription termination with gene expression regulation. SIGNIFICANCE STATEMENTAt the 3 end of genes two processes occur, which are often treated as one: pre-mRNA 3 processing and transcription termination. In fact, they are promoted by a common set of protein factors, with oncogenic properties. Here, we employ a cellular model of colorectal cancer (CRC) progression, to study changes in both pre-mRNA 3 processing and transcription termination. Unexpectedly, we uncover that those changes occur in opposite directions: mRNA tends to be lengthened, while termination is accelerated in cells from primary tumors. We show that the cleavage-termination distance is strongly reduced in these cells compared to normal and metastatic cells, and further determine that cleavage-termination proximity correlates with enhanced gene expression. This uncovers a new layer of gene expression regulation.

molecular biology↗

bioRxiv2025

SETD2 methyltransferase activity aids gene definition by promoting correct transcription initiation and termination

SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma (RCC). In RCC, SETD2 mutations have been linked to delayed transcription termination, but not explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. Majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15-25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity, however widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3' pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and unrelated to transcription readthrough. In summary, we demonstrate that methyltransferase activity of SETD2 regulates transcriptional integrity by stimulating proper initiation, preventing cryptic initiation and promoting efficient 3' end processing.

molecular biology↗

bioRxiv2018

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3 mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

molecular biology↗

bioRxiv2018

The non-canonical SMC protein SmcHD1 antagonises TAD formation on the inactive X chromosome

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), and formation of two mega-domains. In this study we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 null cells revealed the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred in an acute SmcHD1 knockout model, but in this case, independent of Xi gene de-repression. We conclude that SmcHD1 is a key factor in antagonising TAD formation on Xi.

molecular biology↗