Transcriptional activation of the PKLR gene by novel erythroid-specific regulatory elements

Abstract

The pyruvate kinase L/R (PKLR) gene encodes the L- and R-type isoforms of pyruvate kinase, which catalyze the final step of glycolysis in mammals. The L-type isozyme is mainly found in liver cells, whereas the R-type isozyme is produced specifically in erythroid cells. To investigate the transcriptional activation of the PKLR gene for the R-type isozyme, we analyzed chromatin features—including DNase I sensitivity, histone modifications, and enhancer–promoter interactions—in erythroid K562 and non-erythroid HUVEC cells. Putative regulatory elements, including a promoter and two enhancers, were identified near the PKLR locus in K562 cells. Depletion of H3K4me1 and H3K27ac, histone marks associated with active enhancers, through mutation of histone methyltransferases led to a marked reduction in PKLR transcription, while transcription of a nearby gene remained stable. These regulatory elements were highly occupied by the erythroid-specific transcription factors GATA1 and TAL1. Loss of either factors disrupted local H3K27ac and reduced the recruitment of chromatin-looping factors, resulting in decreased PKLR transcription. Furthermore, CRISPR/Cas9-mediated deletion of the putative regulatory elements significantly diminished PKLR transcription, demonstrating their functional importance. Collectively, these findings highlight the essential role of these regulatory elements in activating PKLR transcription in erythroid cells and emphasize the requirement of erythroid-specific factors for their function.