LPP, the miR-28 host gene, is overexpressed in HEL cells through the transcriptional activation of an upstream alternative promoter bound by STAT5. (A) Relative expression of the LPP (LIM domain lipoma-preferred partner) transcript and miR-28 in the indicated cell lines. The LPP level in UT-7 parental and UT-7 JAK2 WT cell lines have been arbitrarily set at 1. (B) 5′-Rapid amplification of cDNA Ends (RACE)–PCR: a nested PCR on cDNA integrating an adaptor sequence at the 5′end of the LPP transcript amplified a 200-bp band in UT-7 and a larger 300- to 400-bp band in HEL cell lines. After sequencing, the 216-bp band amplified from UT-7 contains exon 1 and 2 of the LPP gene. The 300- to 400-bp band amplified from HEL contains 2 alternative exons (A and B) and exon 2 of the LPP gene. (C) A schematic view of the human LPP gene (LIM domain lipoma-preferred partner) represented with its 11 exons (black boxes), the 2 alternative exons (A and B) and the pri-miR-28 stem loop sequence in intron 6. The 2 identified promoters are indicated by arrows and labeled as Prom A (alternative) and Prom 1 (normal start site). (D) Quantitative PCR on indicated targets after chromatin immunoprecipitation (ChIP) relative to the starting DNA quantity before immunoprecipitation (% INPUT ± SD). Gray and black histograms represent ChIP performed on JAK2 WT and V617F UT-7 cells or UT-7 and HEL cells, respectively. White histograms are negative controls. The actin gene amplification (ACTB) was used a negative control for STAT5B binding. Quantitative PCR for RNA pol II ChIP were normalized against a non transcribed region located outside (3′) of the LPP gene.