The HMG Domain Protein SSRP1/PREIIBF Is Involved in Activation of the Human Embryonic β-Like Globin Gene

ABSTRACT

The human embryonic β-like globin (ɛ-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the ɛ-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An ∼85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human ɛ-globin gene.

ACKNOWLEDGMENTS

We are grateful to Humphrey Wattanga for screening the cDNA expression libraries and for assisting with DNA sequencing. We thank Bill Forrester, Bob Kingston, Tom Maniatis, and Ranjan Sen for helpful discussions and Mark Ptashne for DNA constructs. Brian Dynlacht, Bill Forrester, Jun Ma, and Ranjan Sen provided thoughtful comments on the manuscript.

This work was supported by grants to M.H.B. from the National Institutes of Health (RO1 GM42413) and the Lucille P. Markey Charitable Trust (87-24). M.A.D. and P.J.H. were supported in part by NIH predoctoral training grant GM 07598. During the initial stages of this work, M.H.B. was a Lucille P. Markey Scholar in Biomedical Science.