The distinctive roles of erythroid specific activator GATA-1 and NF-E2 in transcription of the human fetal γ-globin genes

GATA-1 and NF-E2 are erythroid specific activators that bind to the β-globin locus. To explore the roles of these activators in transcription of the human fetal stage specific γ-globin genes, we reduced GATA-1 and p45/NF-E2 using shRNA in erythroid K562 cells. GATA-1 or p45/NF-E2 knockdown inhibited the transcription of the γ-globin genes, hypersensitive site (HS) formation in the LCR and chromatin loop formation of the β-globin locus, but histone acetylation across the locus was decreased only in the case of GATA-1 knockdown. In p45/NF-E2 knockdown cells, GATA-1 binding was maintained at the LCR HSs and γ-globin promoter, but NF-E2 binding at the LCR HSs was reduced by GATA-1 knockdown regardless of the amount of p45/NF-E2 in K562 cells. These results indicate that histone acetylation is dependent on GATA-1 binding, but the binding of GATA-1 is not sufficient for the γ-globin transcription, HS formation and chromatin loop formation and NF-E2 is required. This idea is supported by the distinctive binding pattern of CBP and Brg1 in the β-globin locus. Furthermore GATA-1-dependent loop formation between HS5 and 3′HS1 suggests correlation between histone modifications and chromatin looping

Spatial configuration of the chicken α-globin gene domain: immature and active chromatin hubs

The spatial configuration of the chicken α-globin gene domain in erythroid and lymphoid cells was studied by using the Chromosome Conformation Capture (3C) approach. Real-time PCR with TaqMan probes was employed to estimate the frequencies of cross-linking of different restriction fragments within the domain. In differentiated cultured erythroblasts and in 10-day chick embryo erythrocytes expressing ‘adult’ αA and αD globin genes the following elements of the domain were found to form an ‘active’ chromatin hub: upstream Major Regulatory Element (MRE), −9 kb upstream DNase I hypersensitive site (DHS), −4 kb upstream CpG island, αD gene promoter and the downstream enhancer. The αA gene promoter was not present in the ‘active’ chromatin hub although the level of αA gene transcription exceeded that of the αD gene. Formation of the ‘active’ chromatin hub was preceded by the assembly of multiple incomplete hubs containing MRE in combination with either −9 kb DHS or other regulatory elements of the domain. These incomplete chromatin hubs were present in proliferating cultured erythroblasts which did not express globin genes. In lymphoid cells only the interaction between the αD promoter and the CpG island was detected

Nucleosome and transcription activator antagonism at human β-globin locus control region DNase I hypersensitive sites

Locus control regions are regulatory elements that activate distant genes and typically consist of several DNase I hypersensitive sites coincident with clusters of transcription activator binding sites. To what extent nucleosomes and activators occupy these sites together or exclusively has not been extensively studied in vivo. We analyzed the chromatin structure of human β-globin locus control region hypersensitive sites in erythroid cells expressing embryonic and fetal globin genes. Nucleosomes were variably depleted at hypersensitive sites HS1-HS4 and at HS5 which flanks the 5′ of the locus. In lieu of nucleosomes, activators were differentially associated with these sites. Erythroid–specific GATA-1 resided at HS1, HS2 and HS4 but the NF-E2 hetero-dimer was limited to HS2 where nucleosomes were most severely depleted. Histones H3 and H4 were hyperacetylated and H3 was di-methylated at K4 across the LCR, however, the H3 K4 MLL methyltransferase component Ash2L and histone acetyltransferases CBP and p300 occupied essentially only HS2 and the NF-E2 motif in HS2 was required for Ash2L recruitment. Our results indicate that each hypersensitive site in the human β-globin LCR has distinct structural features and suggest that HS2 plays a pivotal role in LCR organization at embryonic and fetal stages of globin gene expression

TMEM8 – a non-globin gene entrapped in the globin web

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs

Quantitative Proteomics Reveals Myosin and Actin as Promising Saliva Biomarkers for Distinguishing Pre-Malignant and Malignant Oral Lesions

Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells.Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early