Regulation of transcription and splicing of pyruvate kinase isozyme genes

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1. We purified nuclear protein bound to the L-II element of pyruvate kinase (PK) L gene and identified them as NF1L and NF1/Red1, members of NF1 family, based upon their partial amino acid sequences. These transcription factors and HNF4 interacted with overlapping sequences in the L-II and affected transcription in an opposite manner. Since NF1 family members also bound to an accessory element of the S14 gene, they are suggested to be involved in carbohydrate regulation of the PKL gene.2. We isolated two cDNA clones interacting with the L-III element of the PKL gene using yeast one-hybrid system and identified them as Hex, a homeobox protein. However, further analysis revealed that Hex was not a L-III binding protein. This protein stimulated acitivities of HNF1 and HNF4, but its physiological significance has never been clarified. Hex was shown to be a transcriptional repressor which may be invloved in differentiation and/or maintenance of differentiated state in hepatocytes.3. The L-III element binding protein (LIIIBP) of the PKL gene was purified from rat liver nuclear extracts. Purified LIIIBP showed two bands of 26 and 24 kDa on SDS/PAGE and different from USF based upon their heat staability and immunoreactivity. UV-crosslinking study revealed that the two proteins specifically bound to the L-III element.4. Of three regulatory region (boxes A, B and C) of the PKM gene, it was found that boxes B and C palyed a major role in transcriptional regulation of the PKM gene. Electrophoretic mibility shift assay revealed that members of Sp1 family such as Sp1 and Sp3 bound to boxes A and B, and that NF-Y bound to Box C.