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B Muralikrishna

Publications and source records attributed to B Muralikrishna.

5 recordsLinked to original sources
PubMed2001

SP3 and AP-1 mediate transcriptional activation of the lamin A proximal promoter.

Lamin A is a major component of the nuclear lamina that is expressed in various types of differentiated cells. We have analysed previously the putative promoter sequences of the gene and shown that the rat lamin A proximal promoter contains two essential motifs, a GC box that can bind to Sp1 and Sp3, and an AP-1 motif that can bind to c-Jun and c-Fos. In this study we have investigated the role of Sp1 and Sp3 in transactivation of the promoter. Functional analysis of the promoter in Drosophila SL2 cells has demonstrated that it is inactive in the absence of Sp proteins. Activation by expression of Sp3 is more pronounced than that by Sp1 although both proteins can bind to the GC box in vitro; activation clearly depends on an intact GC box as deduced from mutant analysis. Promoter activity in SL2 cells also requires an intact AP-1 motif, which can bind to endogenous Drosophila Jun and Fos proteins. Furthermore, overexpression of c-Jun and c-Fos results in fourfold activation of the promoter in PCC-4 embryonal carcinoma cells. Our demonstration that activation of the lamin A proximal promoter is mediated by Sp3 and AP-1 transcription factors affords a basis for further studies on the regulation of this important gene during development and disease.

Animals↗
PubMed2001

Distinct changes in intranuclear lamin A/C organization during myoblast differentiation.

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.

Animals↗
PubMed1999

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Animals↗
PubMed1998

Functional analysis of the 5' promoter region of the rat lamin A gene.

The A-type lamins are constituents of the nuclear lamina in differentiated cells and have been proposed to play an important role in nuclear organization. In this study, we isolated and characterized a genomic clone containing the putative promoter region of the rat lamin A gene. Sequence analysis of about 2 kb of this region combined with primer extension data revealed the presence of a TATA box at -33, a GC box at -101, and AP1 motifs at -7, -424, and -1677. Deletion analysis of the promoter fragments in three mammalian cell lines indicated that a 221-bp segment of the proximal promoter containing the GC box and AP1 motif at -7 was sufficient to give high levels of luciferase activity in reporter gene assays. Mutations in these two motifs resulted in considerable loss of reporter gene activity. Analysis by electrophoretic mobility shift assays (EMSAs) has provided evidence for specific binding of the AP1 and Sp1 family of transcription factors to the promoter, a conclusion supported by DNase I footprinting data. This characterization of the 5' promoter region of the lamin A gene should afford a basis for the further clarification of the mechanism of regulation of this important gene during growth and development.

Animals↗