Browsing by Subject "Transcriptional regulation"

Now showing 1 - 3 of 3

Open Access
Multi-level regulation of even-skipped stripes by the ubiquitous factor Zelda
(The Company of Biologists, 2023-11-23) Önal, Pınar; Bishop, T.R.; Xu, Z.; Zheng, M.; Gunasinghe, M.; Nien, C.Y.; Small, S.; Datta, R.R.
The zinc-finger protein Zelda (Zld) is a key activator of zygotic transcription in early Drosophila embryos. Here, we study Zld dependent regulation of the seven-striped pattern of the pair-rule gene even-skipped (eve). Individual stripes are regulated by discrete enhancers that respond to broadly distributed activators; stripe boundaries are formed by localized repressors encoded by the gap genes. The strongest effects of Zld are on stripes 2, 3 and 7, which are regulated by two enhancers in a 3.8 kb genomic fragment that includes the eve basal promoter. We show that Zld facilitates binding of the activator Bicoid and the gap repressors to this fragment, consistent with its proposed role as a pioneer protein. To test whether the effects of Zld are direct, we mutated all canonical Zld sites in the 3.8 kb fragment, which reduced expression but failed to phenocopy the abolishment of stripes caused by removing Zld in trans. We show that Zld also indirectly regulates the eve stripes by establishing specific gap gene expression boundaries, which provides the embryonic spacing required for proper stripe activation
Open Access
Phosphoproteomic analysis of class IA P110β isoform-specific signal transducers upon PI3K pathway activation
(2023-08) Daloğlu, Beril
PI3K pathway activation is a common event observed in various human cancers. As it regulates cell proliferation, migration and metabolism, it has been widely targeted for anticancer therapies and alteration of drug resistance. Studies focusing on PI3K Class IA isoform-specific inhibition has become critical to achieve alternative targeted treatment methods. Although previous findings show that Class IA isoforms differ in their routes of activation, the downstream targets specific to the isoforms have not been fully profiled yet. This study focuses on the phosphoproteomic analysis of p110 isoform-specific downstream molecules upon PI3K pathway activation. We have created a doxycycline-inducible system in Mouse Embryonic Fibroblasts (MEFs) to express wildtype PIK3CB gene encoding p110β or its catalytic and helical domain over-activating mutants, E1051K and N553S. By immunoblotting, the changes in downstream phosphorylation events were analyzed for MEF p110β mutants or for MEF p110β WT cells upon mitogenic PI3K stimulation. These results were collaborated with the comparative phosphoproteomic data obtained from LC-MS/MS Mass Spectrometry analysis. Our findings show that upon short-term stimulation of the PI3K pathway, proteins associated with transcriptional regulation, cytoskeletal rearrangement, cellular signalling, migration and metabolism are differentially phosphorylated. After longer stimulations on the other hand, proteins involved in cell cycle progression, phosphatase activity, nucleocytoplasmic transport and RNA metabolism become more prominent in the comparative phosphoproteome. Similar to early response proteins in β WT cells, the phosphoproteome of β E1051K cells were associated with cytoskeletal rearrangement and cellular migration. Aligning with the morphological changes observed, proteins involved in anatomical structure regulation were found. Additionally, some tumour suppressors and oncogene proteins were found among the differentially regulated phosphoproteins. On the other hand, the phosphoproteome of β N553S cells were enriched for functions of RNA metabolism, nuclear import and apoptotic regulation. Proteins involved in structural organization and microtubule assembly were also observed. 37 kDa Akt substrate Poly(rC) Binding Protein 1 (Pcbp1) was found as a common differentially phosphorylated protein in the phosphoproteomes of mutant and WT p110β expressing cells. It is an RNA-binding protein associated with metastasis and EMT, and has a critical role in gene expression by coordinating RNA stability and processing. As our results have shown that p110β isoform has a major role in cytoskeletal reorganization, cell migration and transcriptional regulation, studying the regulation of p110β-Pcbp1 axis can pave the way to understand the mechanisms of increased tumour progression in p110β-dependent cells.
Open Access
Protein-DNA dissociation kinetics and chromosome organization in a model bacterial confinement
(2021-09) Koşar, Zafer
Transcriptional initiation and repression require the temporal interactions of transcription factors with DNA. Recent experiments showed that the interaction lifetime is crucial for transcriptional regulation. Relevantly, in vitro single-molecule studies showed that nucleoid-associated proteins (NAPs) dissociate rapidly from DNA through facilitated dissociation (FD) with the increasing phase-solution protein concentration. Nevertheless, it is not clear whether such a concentration-dependent mechanism is functional in bacterial confinement, in which NAP levels and the 3D chromosomal architecture are coupled. Here, we employ extensive coarse-grained molecular simulations, where we model the dissociation of specific and nonspecific dimeric NAPs from a high-molecular-weight circular DNA polymer in a rod-shaped structure constituting the cellular boundaries. Our simulations indicate that the peak cellular protein concentrations result in highly compact chromosomal conformations. Such compactions lead to shorter DNA-residence times but only for NAPs demonstrating sequence-specificity, such as the factor for inversion stimulation (Fis). On the other hand, the dissociation rates of nonspecific NAPs decrease with the increasing protein concentrations, exhibiting an inverse FD behavior. Another set of simulations utilizing restrained chromosome models reveal DNA-segmental fluctuations as the cause of this reversed response, suggesting that moderate chromosomal compaction promotes protein dissociation. Together, our findings suggest that cellular quantities of structural DNA-binding proteins could be highly influential on their residence times and the chromosome architecture.