ABSTRACT
SG2NA is a member of the striatin family of WD-40 repeat proteins with potential scaffolding functions. It was originally identified as a tumor antigen with increased expression during S to G2 phase of cell cycle. We report here that mouse SG2NA has at least five novel splice variants of which two are devoid of the carboxyl terminal WD-40 repeats. The variants of SG2NA are generated by alternative splicing at the exon 7-9 regions and differ in their expression profiles in various tissues tested. While the 83, 78, 38 and 35 kDa variants are present in both brain and heart, the 87 kDa form is brain specific. Also, the expression of 35 kDa variant is more in neonatal than in adult tissues. Western analysis suggests that the SG2NA isoforms differentially respond to growth stimuli. Upon serum stimulation, while the 35 kDa variant is increased, the 78 kDa form is diminished. Splicing variation of SG2NA is conserved in metazoan evolution. In embryonic chicken there are at least four variants of which one is present in brain but absent in heart. Taken together, splicing variation of SG2NA might have some critical roles in differentiation and maturation in metazoan cells.
Subject(s)
Alternative Splicing , Autoantigens/genetics , Calmodulin-Binding Proteins/genetics , Microfilament Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cell Differentiation , Cell Line , Cell Proliferation , Chickens , Humans , Mice , Molecular Sequence Data , Myocardium/metabolism , Organ Specificity , Protein Isoforms/metabolism , RNA SplicingABSTRACT
The family of Ap2 transcription factors comprises five members with highly conserved DNA-binding domains. Among the family members, Ap2delta is the most divergent, because it lacks highly conserved residues within the transactivation domain (TAD) and has weak affinity for known Ap2 binding sites. To identify specific Ap2delta coactivators/regulators during development, we performed a yeast two-hybrid screen, using Ap2delta's TAD. We identified the trithorax superfamily member, Ash2l, as a binding partner that interacts exclusively with Ap2delta. We showed that Ash2l positively mediates Ap2delta transactivation in a dose-dependent manner. Given the known role of Ash2l in histone modification, we determined whether Ap2delta was able to form a complex with that activity. Our results showed that Ap2delta associates with endogenous ASH2L and a member of the MLL family of histone lysine methyltransferases (HKMTs), MLL2 (ALR), forming a complex that methylates lysine 4 of histone H3 (H3K4). Additionally, we showed that Ap2delta is necessary for recruitment of Ash2l and Alr to the Hoxc8 locus and that recruitment of this complex leads to H3K4 trimethylation (H3K4me3) and subsequent gene activation. Altogether, we provide evidence of an association between a highly restricted gene-specific transcription factor and a Su(var), Enhancer of Zeste, Trithorax (SET)1/trithorax-like complex with H3K4 methyltransferase activity. Our studies also document a functional role for Ap2delta in recruiting histone methyltransferases (HMTs) to specific gene targets, such as Hoxc8. This role provides a mechanism through which these transcription factors can have diverse effects despite nearly identical DNA-binding motifs.
Subject(s)
DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Homeodomain Proteins/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Nuclear Proteins/metabolism , Transcription Factor AP-2/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Animals , Cell Line , DNA-Binding Proteins/genetics , Histones/metabolism , Humans , Mice , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
Enormity of the metazoan genomes and divergence in their regulation impose a serious constraint on the comprehensive understanding of context specific gene regulation. DNA elements located in the promoter, enhancer, and other regulatory regions of the genome dictate the temporal and spatial patterns of gene activities. However, owing to the diminutive and variable nature of the regulatory DNA elements, their identification and location remains a major challenge. We have developed an efficient strategy for isolating a repertoire of target sites for sequence specific DNA binding proteins from embryonic chick heart. A comprehensive library of such sequences was constructed and authenticated using various parameters including in silico determination of functional binding sites. This approach, therefore, for the first time, established an experimental and conceptual framework for defining the entire repertoire of functional DNA elements in any cellular context.
