title: Intron 1 GATA site enhances ALAS2 expression indispensably during erythroid differentiation.
abstract: The first intronic mutations in the intron 1 GATA site (int-1-GATA) of 5-aminolevulinate synthase 2 (ALAS2) have been identified in X-linked sideroblastic anemia (XLSA) pedigrees, strongly suggesting it could be causal mutations of XLSA. However, the function of this int-1-GATA site during in vivo development remains largely unknown. Here, we generated mice lacking a 13 bp fragment, including this int-1-GATA site (T AGATAA: AGCCCC) and found that hemizygous deletion led to an embryonic lethal phenotype due to severe anemia resulting from a lack of ALAS2 expression, indicating that this non-coding sequence is indispensable for ALAS2 expression in vivo Further analyses revealed that this int-1-GATA site anchored the GATA site in intron 8 (int-8-GATA) and the proximal promoter, forming a long-range loop to enhance ALAS2 expression by an enhancer complex including GATA1, TAL1, LMO2, LDB1 and Pol II at least, in erythroid cells. However, compared with the int-8-GATA site, the int-1-GATA site is more essential for regulating ALAS2 expression through CRISPR/Cas9-mediated site-specific deletion. Therefore, the int-1-GATA site could serve as a valuable site for diagnosing XLSA in cases with unknown mutations.
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Yingchi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Jingliao
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: An
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wan
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Shihui
authors.initials: S
authors.lastname: Ma
authors.affiliation: Department of Cell Biology, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Immunology, Biochemistry and Molecular Biology, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, China.
authors.forename: Xichuan
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: James Douglas
authors.initials: JD
authors.lastname: Engel
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China shilihongxys@ihcams.ac.cn.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Lihong
authors.initials: L
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China chengtao@ihcams.ac.cn.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.|Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin 300020, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China xfzhu@ihcams.ac.cn.|Division of Pediatric Blood Diseases Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Zhu
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000756:Anemia, Sideroblastic / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D004195:Disease Models, Animal; D004742:Enhancer Elements, Genetic; D041905:Erythroid Cells / Q000166:cytology* / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000378:metabolism*; D005786:Gene Expression Regulation; D005804:Genes, Lethal; D040181:Genetic Diseases, X-Linked / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D057898:Hemizygote; D006801:Humans; D007438:Introns; D020014:K562 Cells; D008297:Male; D009154:Mutation; D010375:Pedigree; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion
chemical_list: D050982:GATA1 Transcription Factor; D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human