Figure 3.
Anemia-activated cis elements are functional E-box–GATA enhancers. (A) Published data of chromatin occupancy and accessibility at the human SSX2IP locus shown in UCSC Genome Browser. Profiles from ChIP-sequencing of H3K27ac (GSM1278239) and TAL1 (GSM1278241) in human bone marrow derived proerythroblasts (ProE), GATA2 (GSM467648) in K562 cells, GATA1 (GSM935465) in human peripheral derived blood-erythroblast (Ery.), and single-cell ATAC-seq peak in human hematopoietic cell types (GSE74310). Light blue line indicates the E-box–GATA sequence. (B) Quantitation of Ssx2ip and Ttll12 mRNA in mouse CD71+ Ter119− spleen cells. Control, untreated WT mouse. Anemia, 3 days after PHZ treatment. (C) FAIRE signal at RPII215, Keratin, Ssx2ip, and Tttl12 loci in mouse mouse CD71+ Ter119− spleen cells. (D) Quantitation of human SSX2IP mRNA and its adjacent genes CTSB and LPAR3 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the SSX2IP + 1.8 (sgSX-E-1 and sgSX-E-2). (E) Quantitation of human TTLL12 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the TTLL12 + 3.4 (sgT12-E-1 and sgT12-E-2). (F) Quantitation of human TRAK2 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the TRAK2 + 3.4 (sgT2-E-1 and sgT2-E-2). (G) Quantitation of human SSX2IP mRNA in GFP+ human umbilical cord blood–derived erythroid progenitor 2 (HUDEP-2) cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron targeting control (sgInt1-NT), and 2 distinct sequences in the TRAK2 + 1.8 (sgSX-E-1 and sgSX-E-2). (H) (Left) Evolutionary conservation of E-box–GATA sequence in mouse, human, and other species. Sequence validation of a CRISPR/Cas9-generated heterozygous deletion in mouse G1E cells. (Right) Allele-specific primary transcript expression at Ssx2ip in heterozygous mouse G1E cells. (I-J) FAIRE signal of CRISPRi in human K562 cells in ACTB-promoter open region control, Ch12 heterochromatin closed region control and (I) SSX2IP E-box–GATA region, SSX2IP promoter region and (J) TTLL12 E-box-GATA region. Enh A and B are 2 sets of primer targeting the same region. Error bars represent SD. ∗P < .05 (two-tailed unpaired Student t test). (K) Quantitative ChIP analysis of GATA1 and TAL1 in K562 cells expressing CRISPRi with sgScrambled or sgSsx2ip-E1 RNAs (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. B, B cell; CD4 T, CD4+ T cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; CRISPRi, CRISPR interference; Ery, Ter119+ erythroid cell; GFP, green fluorescent protein; HSC, hematopoietic stem cells; Mega, megakaryocyte; MEP, megakaryocyte erythroid progenitor; Mono, monocytes; MPP, multipotential progenitors; −RT, no reverse transcriptase; SD, standard deviation; WT, wild-type.

Anemia-activated cis elements are functional E-box–GATA enhancers. (A) Published data of chromatin occupancy and accessibility at the human SSX2IP locus shown in UCSC Genome Browser. Profiles from ChIP-sequencing of H3K27ac (GSM1278239) and TAL1 (GSM1278241) in human bone marrow derived proerythroblasts (ProE), GATA2 (GSM467648) in K562 cells, GATA1 (GSM935465) in human peripheral derived blood-erythroblast (Ery.), and single-cell ATAC-seq peak in human hematopoietic cell types (GSE74310). Light blue line indicates the E-box–GATA sequence. (B) Quantitation of Ssx2ip and Ttll12 mRNA in mouse CD71+ Ter119− spleen cells. Control, untreated WT mouse. Anemia, 3 days after PHZ treatment. (C) FAIRE signal at RPII215, Keratin, Ssx2ip, and Tttl12 loci in mouse mouse CD71+ Ter119− spleen cells. (D) Quantitation of human SSX2IP mRNA and its adjacent genes CTSB and LPAR3 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the SSX2IP + 1.8 (sgSX-E-1 and sgSX-E-2). (E) Quantitation of human TTLL12 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the TTLL12 + 3.4 (sgT12-E-1 and sgT12-E-2). (F) Quantitation of human TRAK2 mRNA in GFP+ K562 cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron control (sgInt1-NT), and 2 distinct sequences in the TRAK2 + 3.4 (sgT2-E-1 and sgT2-E-2). (G) Quantitation of human SSX2IP mRNA in GFP+ human umbilical cord blood–derived erythroid progenitor 2 (HUDEP-2) cells infected with CRISPRi/dCas9-KRAB lentivirus containing sgRNAs targeting Scrambled (sgScrambled), same-intron targeting control (sgInt1-NT), and 2 distinct sequences in the TRAK2 + 1.8 (sgSX-E-1 and sgSX-E-2). (H) (Left) Evolutionary conservation of E-box–GATA sequence in mouse, human, and other species. Sequence validation of a CRISPR/Cas9-generated heterozygous deletion in mouse G1E cells. (Right) Allele-specific primary transcript expression at Ssx2ip in heterozygous mouse G1E cells. (I-J) FAIRE signal of CRISPRi in human K562 cells in ACTB-promoter open region control, Ch12 heterochromatin closed region control and (I) SSX2IP E-box–GATA region, SSX2IP promoter region and (J) TTLL12 E-box-GATA region. Enh A and B are 2 sets of primer targeting the same region. Error bars represent SD. ∗P < .05 (two-tailed unpaired Student t test). (K) Quantitative ChIP analysis of GATA1 and TAL1 in K562 cells expressing CRISPRi with sgScrambled or sgSsx2ip-E1 RNAs (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. B, B cell; CD4 T, CD4+ T cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; CRISPRi, CRISPR interference; Ery, Ter119+ erythroid cell; GFP, green fluorescent protein; HSC, hematopoietic stem cells; Mega, megakaryocyte; MEP, megakaryocyte erythroid progenitor; Mono, monocytes; MPP, multipotential progenitors; −RT, no reverse transcriptase; SD, standard deviation; WT, wild-type.

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