Brr6 plays a role in gene recruitment and transcriptional regulation at the nuclear envelope

Correlation between transcriptional regulation and positioning of genes at the nuclear envelope is well established in eukaryotes, but the mechanisms involved are not well understood. We show that brr6-1, a mutant of the essential yeast envelope transmembrane protein Brr6p, impairs normal positioning and expression of the PAB1 and FUR4- GAL1,10,7 loci. Similarly, expression of a dominant negative nucleoplasmic Brr6 fragment in wild-type cells reproduced many of the brr6-1 effects. Histone chromatin immunoprecipitation (ChIP) experiments showed decreased acetylation at the key histone H4K16 residue in the FUR4-GAL1,10,7 region in brr6-1. Importantly, blocking deacetylation significantly suppressed selected brr6-1 phenotypes. ChIPseq with FLAG-tagged Brr6 fragments showed enrichment at FUR4 and several other genes that showed striking changes in brr6-1 RNAseq data. These associations depended on a Brr6 putative zinc finger domain. Importantly, artificially tethering the GAL1 locus to the envelope suppressed the brr6-1 effects on GAL1 and FUR4 expression and increased H4K16 acetylation between GAL1 and FUR4 in the mutant. Together these results argue that Brr6 interacts with chromatin, helping to maintain normal chromatin architecture and transcriptional regulation of certain loci at the nuclear envelope.

Novel Zinc Finger-Homeodomain Gene from Barley (HvZFHD1) is Differentially Regulated During Spike Development and under Hormonal Treatments and Abiotic Stresses

Plant zinc finger-homeodomains (ZFHDs) are transcriptional factors that play an important role in regulating plant growth and development. Several ZFHD genes were cloned and characterized in many plant species. In the present study, a full-length cDNA sequence of ZFHD gene was cloned from barley (termed as HvZFHD1) using reverse transcription polymerase chain reaction (RT-PCR). The sequence analysis showed that the HvZFHD1 was 1477 bp in length, and contained a complete open reading frame (ORF) of 1161 bp. The deduced protein is composed of 386 amino acids, with a predicted molecular weight of 40.46 kDa and a theoretical isoelectric point of 8.5. Multiple sequence alignment indicated that HvZFHD1 protein shared high identity with ZFHD proteins from wheat, maize, and rice. The predicted HvZFHD1 protein contained the characteristic putative zinc finger domain in the N-terminus and a DNA binding homeodomain in the C-terminus. The expression level of HvZFHD1 was investigated using qRT-PCR during spike development and in response to exogenous phytohormones and abiotic stresses. The results showed that the expression level of HvZFHD1 was fluctuated during spike development with higher expression during anthesis, medium milk, late milk, and early dough stages. The expression of barley ZFHD1 was strongly responsive to abscisic acid treatment and was up-regulated in seedlings treated with methyl jasmonate, salicylic acid, and ethephone. In addition, the expression levels of HvZFHD1 were increased by dehydration, salinity, and heat stress, but not affected by cold stress. The expression patterns of HvZFHD1 suggest that it might play a role in flowering and flower development and is involved in plant responses to abiotic stresses.

MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway

Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.

Four Conserved Cysteine Residues of the Hepatitis B Virus Polymerase Are Critical for RNA Pregenome Encapsidation

ABSTRACT Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.

Conidiophore Stalk-less1 Encodes a Putative Zinc-Finger Protein Involved in the Early Stage of Conidiation and Mycelial Infection in Magnaporthe oryzae

Over recent decades, many pathogenicity genes of Magnaporthe oryzae have been identified but only a very limited number of genes have been identified that encode components of the conidiogenesis pathway. We report here a T-DNA insertional mutant that completely lost conidiation ability. Further investigation revealed that this mutant did not develop any conidiophore, and that the T-DNA was integrated into an annotated gene designated as conidiophore stalk-less1 or COS1. Complementation experiments suggested that COS1 may be a determinant of conidiation. Sequence analysis revealed that COS1 putatively encodes a 491-amino-acid zinc-finger protein and the protein was revealed localized to nucleus. Reverse-transcriptase polymerase chain reaction (RT-PCR)-based expression analysis indicated that two homologues of conidiophore-related genes were affected by the cos1 mutation, suggesting that Cos1 may function as a transcriptional regulator controlling genes responsible for conidiation. Inoculations of rice roots and wounded leaves with mycelia suggested that COS1 is not required for pathogenicity. Moreover, mutation of COS1 may aggravate infection of wounded leaves. Interestingly, different from the wild-type strain, mycelia of the cos1 mutant successfully infected host cells and caused visible symptoms on unwounded leaf blades and sheaths, indicating that Cos1 may have a role in some unknown mechanism of mycelial infection of M. oryzae.