Page 61 - Haematologica March 2020

Genetic and epigenetic regulation of latexin transcription
Such variations in the epigenetic environment result in sig- nificant variations in gene expression, which collectively manifest as a phenotypic trait. Despite these advances, the precise molecular mechanisms underlying the association between the genetic variants and hematopoietic pheno- types remain largely unknown.
Inbred mouse strains provide a model system for explor- ing the myriad of regulatory gene network contributing to hematologic diversity.10,16 We carried out a comparative study of two inbred strains, C57BL/6 (B6) and DBA/2 (D2), in which we documented large natural variation in a number of stem cell traits.5-8 One of the most significant traits is the natural size of the HSC population, i.e. young B6 mice have 3- to 8-fold fewer stem cells in bone marrow (BM) than D2 mice, depending on the assay used for stem cell quantification. We further identified Lxn as the regula- tory gene whose expression is negatively correlated with HSC number.17,18 Lxn regulates HSC in a cell-autonomous manner through concerted mechanisms of decreased self- renewal and increased apoptosis. Even though we identi- fied several genetic variants that might be associated with the differential expression of Lxn in B6 and D2 stem cells, there is no direct evidence of how these variants regulate Lxn transcription and whether they have any functional effects.
In this study, we report for the first time that a chro- matin protein, HMGB2, binds to Lxn promoter and plays an important role in the transcriptional regulation of Lxn. Knockdown of HMGB2 increases Lxn expression at both transcript and protein levels, suggesting a suppressive role of HMGB2 in Lxn transcription. HMGB2 knockdown decreases the number of functional HSC by promoting apoptosis and reducing proliferation. Concomitant knock- down of Lxn reverses these functional effects, suggesting that Lxn is one of the downstream targets of HMGB2. Moreover, we discovered that a functional polymorphism, SNP rs31528793, is associated with the differential expres- sion of Lxn in different mouse strains, including B6 and D2. This study, for the first time, reveals the genetic and epigenetic regulation of Lxn transcription, suggesting that both trans- and cis-elements (HMGB2 and SNP, respec- tively) contribute to the differential gene expression and phenotypic diversity in the HSC population
Methods
Luciferase reporter assay
Lxn promoter activity and HMGB2 transcription activity were measured by luciferase reporter assay with a Tropix TR717 lumi- nometer using a dual luciferase assay kit.
Identification of Lxn promoter binding proteins
Lxn promoter binding proteins were isolated by μMACSTM FactorFinder Kit (Miltenyi Biotec Inc., Auburn, CA, USA). The high purity double-strand DNA oligonucleotides containing SNP rs31528793 was used as the DNA bait for protein pull-down. The associated proteins were determined by mass spectrometry at the Mass Spectrometry and Proteomics Facility at Ohio State
University.
Protein-DNA binding assays
Chromatin immunoprecipitation: chromatin immunoprecipitation (ChIP) assay was performed on LK (Lin- c-KIT+) cells using ChIP assay kit (Sigma Aldrich, #CHP1) with HMGB2 polyclonal anti-
Table 1. Lxn promoter binging protein.
Lxn promoter sequence containing SNP rs31528793
Histone H2A type 1- F
Histone H2A type 2- A Protein S100-A9 Protein S100-A8 Histone H2A.X
High mobility group protein B2 Histone H2B type 1- H Histone H2AV
Histone H1.3
Peptidyl-prolyl cis-trans isomerase A Eosinophil cationic protein 1 precursor Myeloperoxidase precursor
Histone H1.1
Histone H1.5 Coronin-1A
(H2A1F)
(H2A2A)
(S10A9)
(S10A8)
(H2A.X)
(HMGB2)
(H2B1H)
(H2AV)
(H1.3)
(PPIA)
(ECP1)
(PERM)
(H11)
(H15)
(COF1)
Double-strand DNA oligonucleotides containing single nucleotide polymorphism (SNP rs31528793) were used as “bait” to capture associated proteins from bone mar- row cell lysate of C57BL/6 mouse. Proteins binding to Lxn promoter sequences were isolated by μMACSTM FactorFinder Kit (Miltenyi Biotec Inc., Auburn, CA, USA) and identified by Mass Spectrometry.Proteins with a Mascot score of 100 or higher with a minimum of two unique peptides from one protein having a –b or –y ion sequence tag of five residues or better were considered significant.
bodies (ab67282), H2A.X antibody (ab11175), or Rabbit IgG con- trol (ab171870) (Abcam, Cambridge, MA, USA). HMGB2 binding affinity was determined by SYBR green quantitative polymerase chain reaction (qPCR).
Electrophoretic mobility shift assay: electrophoretic mobility shift assays (EMSA) were performed in 293T cells transduced with HMGB2 lentivirus using the LightShiftTM Chemiluminescent EMSA Kit (Thermo ScientificTM).
Gene knockdown and expression measurement
EML or c-KIT+ (LSK) cells were transduced with HMGB2 shRNA (MSH027321-LVRU6GP, GeneCopoeia), Lxn Mission shRNA (Sigma-Aldrich) virus. Gene expression was measured by real-time PCR with commercially available primer/probe mix for Hmgb2 or Lxn in ABI PRISM 7700 (Applied Biosystems, Foster City, CA, USA). Protein expression was measured by western blot with anti-Hmgb2 antibody (ab67282), goat polyclonal anti-Lxn antibodies (ab59521, Abcam), or mouse monoclonal anti-β-actin antibody (A5441, Sigma).
Immunostaining and flow cytometry
Hematopoietic stem cells and hematopoietic progenitor cells: young (8- 12 week) female C57BL/6, DBA2, 129X1/SvJ, A/J and CD45.1 mice (Jackson Laboratories, Bar Harbor, ME, USA) were used for HSC/hematopoietic progenitor cell isolation (HPC). HSC/HPC were defined as Lin–, Sca-1+ (clone E13-161.7) and c-KIT+ (LSK) cells. Long-term HSC (LT-HSC) were identified as LSK plus CD34 and FLT3 negative cells.
Cell cycle: cell cycle was analyzed by BrdU incorporation using BrdU Flow Kit.
Apoptosis: apoptosis was evaluated by Annexin V staining.
Active caspase 3 analysis: active caspase 3 analysis was analyzed using PE Active Caspase-3 Apoptosis Kit. All kits are from BD PharmingenTM. Flow cytometry was performed on a FacsAria II (Becton Dickinson) and the data were analyzed with FlowJo soft- ware (Tree Star, Ashland, OR, USA).
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