Biology subjects

Bakal, C.

Publications and source records attributed to Bakal, C..

4 recordsLinked to original sources

bioRxiv2018

Wdr5, Brca1 and Bard1 link the DNA damage response to the mesenchymal-to-epithelial transition during early reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells (iPSC). We have performed high-content screening with siRNAs targeting 300 chromatin-associated factors. We used colony features, such as size and shape, as well as strength and homogeneity of marker gene expression to define five colony phenotypes in early reprogramming. We identified transcriptional signatures associated with these phenotypes in a secondary RNA sequencing screen. One of these phenotypes involves large colonies and an early block of reprogramming. Double knockdown epistasis experiments of the genes involved, revealed that Brca1, Bard1 and Wdr5 functionally interact and are required for both the DNA damage response and the mesenchymal-to-epithelial transition (MET), linking these processes. Moreover, the data provide a resource on the role of chromatin-associated factors in reprogramming and underline colony morphology as an important high dimensional readout for reprogramming quality.

developmental biology↗

bioRxiv2018

Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

cell biology↗

bioRxiv2018

The dynamics of ERK signaling in melanoma, and the response to BRAF or MEK inhibition, are cell cycle dependent.

Activating BRAF mutations drive melanoma tumorigenesis and metastasis by constitutively activating MEK and ERK, and small molecule inhibitors (SMIs) of BRAF or MEK have shown promise as melanoma therapeutics. However, the development of resistance to these inhibitors in both the short- and longterm is common; warranting investigation into how these SMIs influence ERK signaling dynamics. By quantitative single cell imaging of an ERK activity reporter in living cells, we describe both intra- and inter-cell heterogeneity in ERK activity in isogenic melanoma populations harboring a BRAFV600E mutation. This heterogeneity is largely due to a cell-cycle dependent bifurcation in ERK activation. Moreover, we show there are cell-cycle dependent responses in ERK activity following BRAF or MEK inhibition. Prior to, but not following, CDK4/6-mediated passage through the Restriction Point (RP) ERK activity is sensitive to BRAF and MEK inhibitors. In contrast, in cells that have passed the RP, ERK activity will remain elevated even in the presence of BRAF or MEK inhibition until mitosis. We propose that ERK activity in the presence of activating BRAF mutations is regulated by both positive and negative feedback loops that are engaged in cell-cycle dependent fashions. CDK4/6 inhibition sensitizes ERK activity to BRAF or MEK inhibition by preventing passage the transition from a BRAF/MEK dependent to independent state. Our results have implications for the use of MEK and BRAF inhibitors as melanoma therapeutics, and offer a rational basis for the use of these inhibitors in combination with CDK4/6 inhibition during cancer therapy.

cancer biology↗

bioRxiv2017

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.

genomics↗