Principal Investigator Harvey Lodish
This research project involves determining the transcriptional regulatory networks governing the important changes in gene expression that occur during terminal proliferation and differentiation of erythroid precursors. She began by using chromatin immunoprecipitation with antibodies specific for various erythroid- important transcription factors (ChIP), followed by hybridization of the recovered DNA to a promoter DNA microarray (ChIP-chip). She has since, in collaboration with members of Rick Young’s laboratory, moved onto sequencing of the resulting DNA fragments (ChIP-Seq). This protocol enables her to determine all of the genes that have critical erythroid-important transcription factors bound to their promoter/ enhancer segments. Initial studies focus on transcriptional activation by Stat5, Klf1, and Foxo3, but other factors will soon be investigated. Shilpa's long-term goal is to understand how the complex pattern of gene expression during terminal erythroid differentiation is regulated by transcription factors activated initially by signal transduction pathways downstream of the EpoR, but active in precursors no longer dependent on erythropoietin.
The second tier of this transcriptional network was evaluated by comprehensive mRNA expression profiling during erythroid differentiation: by isolation of mRNA from purified erythroid precursors in successive differentiation stages followed by hybridization to DNA microarrays and eventual confirmation of expression of selected genes by qRT-PCR. In collaboration with Bill Wong, this expression profiling has been confirmed and expanded by second-generation high throughput sequencing (RNA-seq). The results indicate that major changes in gene regulation occur during early erythroblast differentiation, concomitant with induction of Ter119 expression, an erythroid-specific cell surface protein, globin mRNAs, and other factors involved in hemoglobin production. Upregulated genes include many expected categories such as those involved in hemoglobin metabolism, heme and porphyrin ring metabolism, cell and nuclear membrane structure, iron homeostasis, negative regulators of cell cycle, oxygen transport, and metabolism of oxygen and reactive oxygen species. Genes that were significantly downregulated included those involved in TNF-alpha production, NADP metabolism, NF-kappaB binding, actin binding, ubiquitin protein ligation, and non-erythroid specific functions such as immune responses and phagocytosis.
Along with her technical assistant, Karly Burke, Shilpa studied the effects of a specific kinase, Hipk2, which modulates the function of other transcription factors and cofactors and chromatin-modifying enzymes. Hipk2 is highly induced during primary mouse fetal liver erythropoiesis and specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation – probably explained by cell cycle arrest as well as increased apoptosis – and terminal enucleation as well as the reduced accumulation of hemoglobin. Hipk2 knockdown reduces the expression of some genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, but does not affect the induction of several erythroid-specific transcription factors. This suggests that Hipk2 plays a significant role in terminal fetal liver erythroid differentiation and may regulate hemoglobin expression through noncanonical regulatory pathways.
Recently, along with her technical assistant Jennifer Eng, Shilpa has begun to uncover an unusual regulator of erythroid development, specifically chromatin condensation and enucleation, the nuclear export protein, Xpo7. Xpo7 is highly erythroid specific, abundant, and regulated by some of the master erythroid regulators. It is unusual for an export protein in that it does not require a specific nuclear export signal as do all other export proteins. Interestingly, all other nuclear export protein transcripts are repressed during terminal erythropoiesis except for Xpo7. Shilpa studied the function of Xpo7 by shRNA knockdown and discovered that erythroblast nuclei from Xpo7-kd cells were less condensed and larger than control nuclei (by confocal immunofluorescence microscopy). Xpo7-KD nuclei also retained almost all nuclear proteins while normal extruded nuclei had very little protein, as judged both by silver stained gels and mass spectrometry, suggesting that perhaps Xpo7 is a nonspecific nuclear export protein that removes all nuclear protein from the erythroid nucleus in order to allow chromatin to condense.
mRNA-seq analysis accurately quantifies the absolute abundance of individual genes and also the fold changes at different developmental changes. Some of the abundantly expressed genes, in particular transcription factors such as GATA1, Sp2, FOG1 and LMO2, show less than 2 fold induction during erythroid differentiation, yet they are critical for erythropoiesis. Another class of highly expressed genes shows more than 10 fold induction during erythropoiesis, including hbb-b1, hbb-b2, Alas2, Band3, Darc, and Tmod1. Examination of abundant induced genes, which were not previously implicated in erythroid development, identified a number of novel stress hormone related receptors, transcription factors, serine/threonine kinases, non-coding RNAs and splice variants. Together two UROPs, Paula Trepman and Katherine Luo, Bill is determining the functions or many of these new proteins, focusing on the mechanism by which several splice variants are formed and the functions of the different isoforms of several of these erythroid- induced proteins. Their functions are being studied by knocking down their expression in fetal liver erythroid CFU-E progenitors using shRNAs and chemical inhibitors and examining erythroid proliferation and differentiation in culture.