Biology subjects

Kamakaka, R. T.

Publications and source records attributed to Kamakaka, R. T..

5 recordsLinked to original sources

bioRxiv2023

Yeast Heterochromatin Only Stably Silences Weak Regulatory Elements by Altering Burst Duration

The interplay between nucleosomes and transcription factors leads to programs of gene expression. Transcriptional silencing involves the generation of a chromatin state that represses transcription and is faithfully propagated through DNA replication and cell division. Using multiple reporter assays, including directly visualizing transcription in single cells, we investigated a diverse set of UAS enhancers and core promoters for their susceptibility to heterochromatic gene silencing. These results show that heterochromatin only stably silences weak and stress induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements and the partial repression did not result in bistable expression states. Permutation analysis of different UAS enhancers and core promoters indicate that both elements function together to determine the susceptibility of regulatory sequences to repression. Specific histone modifiers and chromatin remodellers function in an enhancer specific manner to aid these elements to resist repression suggesting that Sir proteins likely function in part by reducing nucleosome mobility. We also show that the strong housekeeping regulatory elements can be repressed if silencer bound Sir1 is increased, suggesting that Sir1 is a limiting component in silencing. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating type gene regulatory elements but not strong housekeeping gene regulatory sequences which could help explain why these genes are often found at the boundaries of silenced domains.

molecular biology↗

bioRxiv2021

A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

Dysregulation of the RNA polymerase III (Pol III) transcription program, which synthesizes tRNA and other classes of small noncoding RNA critical for cell growth and proliferation, is associated with cancer and human disease. Previous studies have identified two distinct Pol III isoforms defined by the incorporation of either subunit POLR3G (RPC7) during early development, or POLR3GL (RPC7{beta}) in specialized tissues. Though POLR3G is re-established in cancer and immortalized cell lines, the contributions of these isoforms to transcription potential and transcription dysregulation in cancer remain poorly defined. Using an integrated Pol III genomic profiling approach in combination with in vitro differentiation and subunit disruption experiments, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. In particular, we observe that a specific class of small noncoding RNA, SNAR-A, is exquisitely sensitive to the availability of subunit POLR3G in proliferating cells. Taken further, large-scale analysis of Pol III subunit expression and downstream chromatin features identifies concomitant loss of POLR3G and SNAR-A activity across a multitude of differentiated primary immune cell lineages, and conversely, coordinate re-establishment of POLR3G expression and SNAR-A features in a variety of human cancers. These results altogether argue against strict redundancy models for subunits POLR3G and POLR3GL, and instead support a model in which Pol III identity itself functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.

molecular biology↗

bioRxiv2020

Quantitative analysis of Histone modifications in gene silencing

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. This transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown and we developed a method to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state. We show that loss of silencing required between 50% and 75% of the unacetylated histones to be replaced with acetylated histone mimics. The precise levels of unacetylated nucleosomes required varied from locus to locus and was influenced by both silencer strength and UAS enhancer/promoter strength. Simple calculations suggest that an approximately 50% reduction in the ability of acetylases to acetylate individual nucleosomes across a large domain may be sufficient to generate a transcriptionally silent region in the nucleus.

molecular biology↗

bioRxiv2019

Combinatorial analysis of Saccharomyces cerevisiae regulatory elements

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin components has led to the definition and delineation of various regulatory elements. Enhancers, promoters, 5 untranslated regions (5UTR) and transcription terminators/3 untranslated regions (3UTR) have all been defined. However, the specific contributions of each of these features as part of a regulatory unit and the functional communications between these regulatory elements remains under explored.\n\nWe built a combinatorial library of 26 different enhancers, core promoters, 5UTRs and transcription terminators/3UTRs. This library was analyzed with respect to gene expression in order to better understand the interactions between different regulatory elements. In the process we developed new methods to estimate the contribution of individual regulatory parts from just a few simple measurements. Our data show that different pairs of regulatory parts follow specific interaction rules affecting overall activity either positively or negatively. We find that while enhancers are the initiators of gene activity, core promoters modulate the levels of enhancer mediated expression. Cluster analysis based on expression show that TATA-box containing core promoters appear to increase enhancer-driven transcription to a greater extent than TATA-less promoters. Principal component analysis highlight outliers and suggest differences in mechanisms of regulation. These results provide a system to characterize regulatory elements and use these elements in the design of synthetic regulatory circuits.

synthetic biology↗

bioRxiv2018

Transfer RNA Genes Affect Chromosome Structure and Function via Local Effects

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.

molecular biology↗