AB4138 Sigma-AldrichAnti-KLF4 Antibody

Functional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.

Human induced pluripotent stem (iPS) cells have great value for regenerative medicine, but are facing problems of low efficiency. MicroRNAs are a recently discovered class of 19-25 nt small RNAs that negatively target mRNAs. miR302/367 cluster has been demonstrated to reprogram mouse and human somatic cells to iPS cells without exogenous transcription factors, however, the repetition and differentiation potentiality of miR302/367-induced pluripotent stem (mirPS) cells need to be improved. Here, we showed overexpression of miR302/367 cluster reprogrammed human embryonic kidney 293T cells into mirPS cells in serum-free N2B27-based medium. The mirPS cells had similar morphology with embryonic stem cells, and expressed pluripotent markers including Oct4, Sox2, Klf4, and Nanog. In addition, through formation of embryoid bodies, various cells and tissues from three germ layers could be determined. Moreover, we examined the potential of mirPS cells differentiating into germ cells both in vitro and in vivo. Taken together, these data might provide a new source of cells and technique for the investigation of the mechanisms underlying reprogramming and pluripotency.

Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells.