Jong Joo Lee

The role of transcriptional activator GATA-1 at human β-globin HS2

GATA-1 is an erythroid activator that binds β-globin gene promoters and DNase I hypersensitive sites (HSs) of the β-globin locus control region (LCR). We investigated the direct role of GATA-1 interaction at the LCR HS2 enhancer by mutating its binding sites within minichromosomes in erythroid cells. Loss of GATA-1 in HS2 did not compromise interaction of NF-E2, a second activator that binds to HS2, nor was DNase I hypersensitivity at HS2 or the promoter of a linked ε-globin gene altered. Reduction of NF-E2 using RNAi confirmed the overall importance of this activator in establishing LCR HSs. However, recruitment of the histone acetyltransferase CBP and RNA pol II to HS2 was diminished by GATA-1 loss. Transcription of ε-globin was severely compromised with loss of RNA pol II from the transcription start site and reduction of H3 acetylation and H3K4 di- and tri-methylation in coding sequences. In contrast, widespread detection of H3K4 mono-methylation was unaffected by loss of GATA-1 in HS2. These results support the idea that GATA-1 interaction in HS2 has a prominent and direct role in co-activator and pol II recruitment conferring active histone tail modifications and transcription activation to a target gene but that it does not, by itself, play a major role in establishing DNase I hypersensitivity.

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

Zebrafish Jak2a plays a crucial role in definitive hematopoiesis and blood vessel formation

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2(-/-) transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.

Corepressor MMTR/DMAP1 Is Involved in both Histone Deacetylase 1- and TFIIH-Mediated Transcriptional Repression

ABSTRACT A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMAP1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase 1 (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase II largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.