Nhp6 Protein Research Articles

Role of the Nhp6 Protein in Nucleosome Unfolding by the FACT Factor

Role of the Nhp6 Protein in Nucleosome Unfolding by the FACT Factor

Role of the Nhp6 Protein in In Vitro Transcription through the Nucleosome

Nhp6 is a small yeast protein that binds DNA nonspecifically. It has been shown that Nhp6 is a component of several protein complexes (including the FACT complex) and is present on many yeast promoters and transcribed regions of genes in vivo. It also participates in the process of destabilizing the structure of nucleosomes in vitro. In our laboratory, we studied the FACT complex and showed its role in transcription by eukaryotic RNA polymerase 2 in vitro. However, the role of the Nhp6 protein in transcription has not been studied previously. In this paper, we describe the effect of the Nhp6 protein on transcription through the nucleosome by eukaryotic RNA polymerase 2 and show that the Nhp6 protein increases the transcription efficiency at several positions on nucleosomal DNA, primarily the transcription at positions +(11–17) in the nucleosome. We proposed a model of the Nhp6 action during transcription through chromatin. The model suggests the stabilization of transient DNA uncoiling from the octamer during this process.

A novel role for Nhp6 proteins in histone gene regulation in Saccharomyces cerevisiae

Maintaining a stable and balanced histone pool is of paramount importance for genome stability and fine regulation of DNA replication and transcription. This involves a complex regulatory machinery, exploiting transcription factors as well as histone chaperones, chromatin remodelers and modifiers. The functional details of this machinery are as yet unclear. Previous studies report histone decrease in mammalian and yeast HMGB family mutants. In this study we find that Nhp6 proteins, the S. cerevisiae HMGB1 homologues, control histone gene expression by affecting nucleosome stability at regulative regions of the histone clusters. In addition, we observe that histone gene overexpression in the nhp6ab mutant is accompanied by downregulated translation, which in turn is responsible for the histone decrease phenotype. Our observations allow us to incorporate Nhp6 proteins into the large group of chromatin factors that tightly regulate histone gene expression.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates

SummaryThe integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.

Variant histone H2A.Z, but not the HMG proteins Nhp6a/b, is essential for the recruitment of Swi/Snf, Mediator, and SAGA to the yeast GAL1 UAS G

Chromatin architecture is very important for the regulation of transcriptional activation. Here, we investigated the role of two different chromatin components, the histone variant H2A.Z and HMG proteins Nhp6a/b, in regulating GAL1 gene expression. We have shown that recruitment of the Mediator complex is significantly affected in the absence of H2A.Z. Furthermore, H2A.Z is also required to fully recruit the SAGA and Swi/Snf complexes to the yeast GAL1-10 UAS G. On the other hand, the HMG protein Nhp6a/b is not required to recruit the aforementioned components to the GAL1 promoter. The Nhp6 protein has been shown to interact with nucleosomes, and we show that its distribution is unaffected in the absence of H2A.Z. Our results suggest that the incorporation of the histone variant H2A.Z, but not the HMG proteins Nhp6a/b, in promoter regions creates a specialized chromatin domain that is required for pre-initiation complex assembly at the GAL1 locus.

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

A key event in tRNA gene (tDNA) transcription by RNA polymerase (Pol) III is the TFIIIC-dependent assembly of TFIIIB upstream of the transcription start site. Different tDNA upstream sequences bind TFIIIB with different affinities, thereby modulating tDNA transcription. We found that in the absence of Nhp6 proteins, the influence of the 5'-flanking region on tRNA gene transcription is dramatically enhanced in Saccharomyces cerevisiae. Expression of a tDNA bearing a suboptimal TFIIIB binding site, but not of a tDNA preceded by a strong TFIIIB binding region, was strongly dependent on Nhp6 in vivo. Upstream sequence-dependent stimulation of tRNA gene transcription by Nhp6 could be reproduced in vitro, and Nhp6 proteins were found associated with tRNA genes in yeast cells. We also show that both transcription and silencing barrier activity of a tDNA(Thr) at the HMR locus are compromised in the absence of Nhp6. Our data suggest that Nhp6 proteins are important components of Pol III chromatin templates that contribute both to the robustness of tRNA gene expression and to positional effects of Pol III transcription complexes.

Human SSRP1 Has Spt16-dependent and -independent Roles in Gene Transcription

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were approximately 170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 ( approximately 1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells.

Structural features of nucleosomes reorganized by yeast FACT and its HMG box component, Nhp6.

The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT.nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.

Gene dosage affects the expression of the duplicated NHP6 genes of Saccharomyces cerevisiae

Nhp6Ap and Nhp6Bp, which are 87% identical in sequence, are moderately abundant, chromosome-associated proteins from Saccharomyces cerevisiae. In wild type cells Nhp6Ap is present at three times the level of Nhp6Bp. The effects of altering NHP6A or NHP6B gene number on the expression of its partner has been examined using Northern blots and reporter genes. Deletion of NHP6A led to a three-fold increase in NHP6B synthesis while an extra copy of NHP6A reduced NHP6B expression two-fold. Changes in the NHP6B gene copy number caused more moderate changes in NHP6A synthesis. The regulation of one NHP6 gene by the other uses a mechanism that detects the level of Nhp6 protein (or RNA) rather than gene number, since overexpression of Nhp6B protein from a single gene led to a dramatic decrease in NHP6A synthesis. Deletion analysis showed that the regulatory element involved in gene dosage compensation maps to a 190 bp segment in the NHP6B promoter. The simplest model, that each Nhp6 protein can act as a transcriptional repressor at the other NHP6 gene, is not true since purified Nhp6A protein does not bind specifically to the NHP6B promoter region. Instead, Nhp6p appears to interact with or through another protein in regulating transcription from the NHP6 genes.

A bipartite yeast SSRP1 analog comprised of Pob3 and Nhp6 proteins modulates transcription.

The FACT complex of vertebrate cells, comprising the Cdc68 (Spt16) and SSRP1 proteins, facilitates transcription elongation on a nucleosomal template and modulates the elongation-inhibitory effects of the DSIF complex in vitro. Genetic findings show that the related yeast (Saccharomyces cerevisiae) complex, termed CP, also mediates transcription. The CP components Cdc68 and Pob3 closely resemble the FACT components, except that the C-terminal high-mobility group (HMG) box domain of SSRP1 is not found in the yeast homolog Pob3. We show here that Nhp6a and Nhp6b, small HMG box proteins with overlapping functions in yeast, associate with the CP complex and mediate CP-related genetic effects on transcription. Absence of the Nhp6 proteins causes severe impairment in combination with mutations impairing the Swi-Snf chromatin-remodeling complex and the DSIF (Spt4 plus Spt5) elongation regulator, and sensitizes cells to 6-azauracil, characteristic of elongation effects. An artificial SSRP1-like protein, created by fusing the Pob3 and Nhp6a proteins, provides both Pob3 and Nhp6a functions for transcription, and competition experiments indicate that these functions are exerted in association with Cdc68. This particular Pob3-Nhp6a fusion protein was limited for certain Nhp6 activities, indicating that its Nhp6a function is compromised. These findings suggest that in yeast cells the Cdc68 partners may be both Pob3 and Nhp6, functioning as a bipartite analog of the vertebrate SSRP1 protein.

Mutations in the Yeast Nhp6 Protein Can Differentially Affect Its in Vivo Functions

Nhp6A and Nhp6B from S. cerevisiae are required for viability at 38°C because they are involved in transcription of SNR6 by RNA polymerase III. Nhp6A also represses transcription of NHP6B by RNA polymerase II. Nhp6 belongs to the HMG1 family, defined by an 80 amino acid DNA binding domain, which includes six highly conserved residues. These amino acids were mutated in Nhp6A and their affects on Nhp6 function were assessed in vivo. Surprisingly, most of the changes allowed Nhp6A to function normally in supporting growth at 38°C. However, six mutants had differential effects on in vivo function. Finally, two of the mutant proteins that did not restore Nhp6A function in vivo were shown to bind and bend DNA in vitro as well as wild type. Together, these results suggest that Nhp6 interacts with another protein(s) to carry out some of its biological functions and that this interaction might differ at promoters transcribed by RNA polymerase II versus RNA polymerase III.

The mitochondrial histone HM: An evolutionary link between bacterial HU and nuclear HMG1 proteins

The mitochondrial histone HM is a very abundant protein in yeast mitochondria that wraps DNA and activates transcription in vitro and is required within the cell for proper maintenance of the mitochondrial chromosome. HM and the bacterial histone-like protein HU have similar activities in vitro and can substitute for each other in E coli cells and in yeast mitochondria. HM also appears to be functionally homologous to nuclear HMG1 proteins, with which it shares a high degree of sequence homology. We report here the isolation of extragenic suppressors of the yeast HM mutant temperature-sensitive phenotype. We also examined the effects of the lack of HM protein and of respiration deficiency on yeast cells mutant for the NHP6 proteins, the putative yeast nuclear HMG1 homologues.

Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1.

The nonhistone chromosomal protein NHP6 from Saccharomyces cerevisiae has been previously isolated and its amino-terminal sequence determined. In this report, synthetic oligonucleotides, designed from the limited NHP6 amino acid sequence, were used as hybridization probes to clone the NHP6A gene from a yeast genomic library. Low stringency Southern blot analysis showed that there was a second gene homologous to NHP6A. This gene, NHP6B, was also cloned and sequenced. Nucleotide sequence analysis revealed that NHP6B has six extra amino acids at its amino terminus, but that NHP6A and NHP6B match at 87% of the rest of their sequences. S1 nuclease analysis was used to show that both genes are transcribed; the major transcription start sites lie 30 bases before the first ATG codon. Interestingly, the approximately 11-kDa NHP6A and NHP6B proteins are homologous to the middle segment of the 27-kDa chromatin-associated high mobility group protein 1 from calf; NHP6A and NHP6B each have over 40% identity with this part of high mobility group protein 1. Possible functions for the NHP6 proteins are discussed in light of this homology.