Biology subjects

Rine, J.

Publications and source records attributed to Rine, J..

12 recordsLinked to original sources

bioRxiv2023

Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells

Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells from which data-rich metrics can be extracted. SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 m field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 m2 /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution.

biophysics↗

bioRxiv2023

Two-way feedback between chromatin compaction and histone modification state explains S. cerevisiae heterochromatin bistability

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. Here, we propose a broader view: chromatin compaction can be both a cause and a consequence of the histone modification state, and this tight bidirectional interaction can underpin bistable transcriptional states. To test this theory, we developed a mathematical model for the dynamics of the HMR locus in S. cerevisiae, that incorporates activating histone modifications, silencing proteins and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states, and vice versa, and protein binding levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. This bidirectional feedback between chromatin compaction and the histone modification state may be an important regulatory mechanism at many loci. SignificanceChromatin is the complex formed by proteins, including histones, and DNA to form chromosomes. Specific chromatin structures and states are thought to be key factors regulating transcription. A common view proposes that histone modifications activate or inhibit transcription either via specific activation or inhibition of RNA polymerase binding/elongation at a locus, or by expanding/compacting the locus, thereby modulating its accessibility to many macromolecules. In this work, we elucidated a broader hypothesis that chromatin compaction may both inhibit transcription, and feedback via silencing proteins to remove histone modifications that further control chromatin compaction and correlate with gene activity. We developed a model incorporating these ideas and showed that it explains quantitative experimental data for a silent locus in budding yeast.

systems biology↗

bioRxiv2023

Context dependent function of the transcriptional regulator Rap1 in gene silencing and activation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Rap1, and Sir proteins. Despite a vast understanding of the establishment and maintenance of Sir-silenced chromatin, the mechanism of gene silencing by Sir proteins has remained a mystery. Utilizing high resolution chromatin immunoprecipitation, we found that Rap1, the native activator of the bi-directional HML promoter, bound its recognition sequence in silenced chromatin and its binding was enhanced by the presence of Sir proteins. In contrast to prior results, various components of transcription machinery were not able to access HML in the silenced state. These findings disproved the long-standing model of indiscriminate steric occlusion by Sir proteins and led to investigation of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a novel role for promoter-bound Rap1 in the maintenance of silent chromatin through interactions with the Sir complex. We also found that promoter-bound Rap1 activated HML when in an expressed state, and aided in the transition from transcription initiation to elongation. Highlighting the importance of epigenetic context in transcription factor function, these results point toward a model in which the duality of Rap1 function was mediated by local chromatin environment rather than binding-site availability. Significance StatementThe coarse partitioning of the genome into regions of active euchromatin and repressed heterochromatin is an important, and conserved, level gene expression regulation in eukaryotes. Repressor Activator Protein (Rap1) is a transcription factor that promotes the activation of genes when recruited to promoters, and aids in the establishment of heterochromatin through interactions with silencer elements. Here, we investigate the role of Rap1 when bound to a promoter in silent chromatin and dissect the context-specific epigenetic cues that regulate the dual properties of this transcription factor. Together, our data highlight the importance of protein-protein interactions and local chromatin state on transcription factor function.

molecular biology↗

bioRxiv2022

Distinct silencer states determine epigenetic states of heterochromatin

A remarkable property of heterochromatin is that a given heterochromatic locus can exhibit different transcriptional states in genetically identical cells. Studies demonstrate that epigenetic inheritance of the silenced state requires silencers and therefore cannot be driven by the inheritance of modified histones alone. To address these observations, we surveyed the chromatin architectures of strong and weak silencers in Saccharomyces. We found that strong silencers recruited Sir proteins and silenced the locus in all cells. Strikingly, weakening these silencers reduced Sir protein recruitment and stably silenced the locus in some cells; however, this silenced state could probabilistically convert to an expressed state that lacked Sir protein recruitment. Additionally, changes in the constellation of silencer-bound proteins or the concentration of a structural Sir protein modulated the probability that a locus existed in the silenced or expressed state. These findings argued that distinct states of silencers generate epigenetic states and regulate their dynamics.

cell biology↗

bioRxiv2021

Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah{Delta}, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3 overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

molecular biology↗

bioRxiv2020

A novel allele of SIR2 reveals a heritable intermediate state of gene silencing

Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1{Delta}, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.

genetics↗

bioRxiv2020

S-phase independent silencing establishment in Saccharomyces cerevisiae

The establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using high-resolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that the cell-cycle-regulated step was downstream of Sir protein recruitment. In contrast to prior results, HML and HMR had identical cell-cycle requirements for silencing establishment, with no apparent contribution from a tRNA gene adjacent to HMR. We identified the cause of the S-phase requirement for silencing establishment: removal of transcription-favoring histone modifications deposited by Dot1, Sas2, and Rtt109. These results revealed that silencing establishment was absolutely dependent on the cell-cycle-regulated interplay between euchromatic and heterochromatic histone modifications.

genetics↗

bioRxiv2019

Epigenetic memory independent of symmetric histone inheritance

Heterochromatic gene silencing is an important form of gene regulation that usually requires specific histone modifications. A popular model posits that inheritance of modified histones, especially in the form of H3-H4 tetramers, underlies inheritance of heterochromatin. Because H3-H4 tetramers are randomly distributed between daughter chromatids during DNA replication, rare occurrences of asymmetric tetramer inheritance within a heterochromatic domain would have the potential to destabilize heterochromatin. This model makes a prediction that shorter heterochromatic domains would experience unbalanced tetramer inheritance more frequently, and thereby be less stable. In contrast to this prediction, we found that shortening a heterochromatic domain in Saccharomyces had no impact on the strength of silencing nor its heritability. Additionally, we found that replisome mutations that disrupt inheritance of H3-H4 tetramers had only minor effects on heterochromatin stability. These findings suggest that histones carry little or no memory of the heterochromatin state through DNA replication.

molecular biology↗

bioRxiv2019

POL30 alleles in Saccharomyces cerevisiae reveal complexities of the cell cycle and ploidy on heterochromatin assembly

In Saccharomyces cerevisiae, transcriptional silencing at HML and HMR maintains mating-type identity. The repressive chromatin structure at these loci is replicated every cell cycle and must be re-established quickly to prevent transcription of the genes at these loci. Mutations in a component of the replisome, the Proliferating Cell Nuclear Antigen (PCNA), encoded by POL30, cause a loss of transcriptional silencing at HMR. We used an assay that captures transient losses of silencing at HML and HMR to perform extended genetic analyses of the pol30-6, pol30-8, and pol30-79 alleles. All three alleles destabilized silencing only transiently and only in cycling cells. Whereas pol30-8 caused loss of silencing by disrupting the function of Chromatin Assembly Factor 1 (CAF-I), pol30-6 and pol30-79 acted through a separate genetic pathway but one still dependent on histone chaperones. Surprisingly, the silencing-loss phenotypes depended on ploidy but not on POL30 dosage or mating-type identity. Separately from silencing loss, the pol30-6 and pol30-79 alleles also displayed high levels of mitotic recombination in diploids. These results established that histone trafficking involving PCNA at replication forks is crucial to the maintenance of chromatin state and genome stability during DNA replication. They also raised the possibility that increased ploidy may protect chromatin states when the replisome is perturbed.

genetics↗

bioRxiv2019

Accumulation of Rare Coding Variants in Genes Implicated in Risk of Human Cleft Lip with or without Cleft Palate

Cleft lip with/without cleft palate (CLP) is a common craniofacial malformation with complex etiologies, reflecting both genetic and environmental factors. Most of the suspected genetic risk for CLP has yet to be identified. To further classify risk loci and estimate the contribution of rare variants, we sequenced the exons in 49 candidate genes in 323 CLP cases and 211 non-malformed controls. Our findings indicated that rare, protein-altering variants displayed markedly higher burdens in CLP cases at relevant loci. First, putative loss-of-function mutations (nonsense, frameshift) were significantly enriched among cases: 13 of 323 cases (~4%) harbored such alleles within these 49 genes, versus one such change in controls (p = 0.01). Second, in gene-level analyses, the burden of rare alleles showed greater case-association for several genes previously implicated in cleft risk. For example, BHMT displayed a 10-fold increase in protein-altering variants in CLP cases (p = 0.03), including multiple case occurrences of a rare frameshift mutation (K400fs). Other loci with greater rare, coding allele burdens in cases were in signaling pathways relevant to craniofacial development (WNT9B, BMP4, BMPR1B) as well as the methionine cycle (MTRR). We conclude that rare coding variants may confer risk for isolated CLP.

genetics↗

bioRxiv2018

A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase

Success in precision medicine depends on our ability to determine which rare human genetic variants have functional effects. Classical homocystinuria--characterized by elevated homocyst(e)ine in plasma and urine--is caused by primarily-rare variants in the cystathionine beta-synthase (CBS) gene. About half of patients respond to vitamin B6 therapy. With early detection in newborns, existing therapies are highly effective. Functional CBS variants, especially those that respond to vitamin B6, can be detected based on their ability to restore growth in yeast cells lacking CYS4 (the yeast ortholog of CBS). This assay has previously been carried out only reactively after first observation of a variant in patients. Here we describe a proactive comprehensive missense variant effect map for human CBS. Together, saturation codon-replacement mutagenesis, en masse growth selection at different vitamin B6 levels, and sequencing yielded a look-up table for CBS missense variant function and vitamin B6-remediability in yeast. The CBS variant effect map identified disease variants and predicted both disease severity (r = 0.82) and human clinical response to vitamin B6 (r = 0.89). Thus, highly-multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

genetics↗

bioRxiv2017

Pivotal roles of PCNA loading and unloading on heterochromatin function

In Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally-silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the PCNA unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.\n\nSIGNIFICANCE STATEMENTDNA replication poses a unique logistical challenge for the cell in that structural features of chromatin and their regulatory functions must be carefully coordinated with passage of replication machinery so faithful duplication of both the genome and its chromatin structures may be achieved. Nucleosome assembly is fundamental to reestablishment of chromatin in the wake of DNA replication, and here a mechanism by which nucleosome assembly is coordinated with DNA replication to maintain silenced chromatin is described.

genetics↗