1. Hmgb3: an HMG-box family member expressed in primitive hematopoietic cells that inhibits myeloid and B-cell differentiation
by Michael J. Nemeth, David J. Curtis, Martha R. Kirby, Lisa J. Garrett-Beal, Nancy E. Seidel, Amanda P. Cline, and David M. Bodine
Blood
Volume 102(4):
August 15, 2003
©2003 by American Society of Hematology

2. Hmgb3 mRNA levels in adult mouse tissues.
Hmgb3 mRNA levels in adult mouse tissues. (A) Northern blot analysis of Hmgb3 expression in adult mouse tissues. Top: Northern blot analysis of Hmgb3 expression in MEL, 32D cell lines, and CTLLs (left) and in adult mouse bone marrow (BM), spleen (Sp), thymus (Ty), brain (Br), heart (He), Liver (Li), Lung (Lu), and Kidney (Ki) (right). Signals corresponding to Hmgb3 mRNA and location of 18s and 28s rRNAs are indicated by arrows. The 3′ untranslated region was used as a probe (yellow bar, Figure 3, middle panel). Bottom: analysis of RNA loading and integrity. 15 μg RNA was loaded onto an agarose gel and stained with ethidium bromide following electrophoresis. (B) RT-PCR analysis of adult mouse bone marrow populations. RT-PCR analysis of bone marrow populations sorted by lineage: lineage-depleted cells, erythroid cells, platelets/megakaryocytes, granulocytes, monocytes/macrophages, mast cells, B cells, T cells, and whole bone marrow. Signals corresponding to Hmgb3 expression are indicated by black arrows. RT-PCR performed without RT (–RT) was used for a negative control. MEL RNA served as a positive control for Hmgb3 expression. β2-microglobulin (β2) was used for an internal control. (C) Semiquantitative duplex RT-PCR analysis of whole, Lin–, c-kit+, Sca-1–(L–K–S–), and Lin–, c-kit+, Sca-1+, IL-7Rα– (L–K+S+I–) bone marrow cells. Hmgb3 mRNA levels were quantified within the linear range of amplification by densitometry and normalized to β2-microglobulin expression. Amplicon sizes are 359 bp and 258 bp for Hmgb3 and β2-microglobulin, respectively. (D) Hmgb3 mRNA in situ hybridization analysis. All analyses were performed on adult mouse lineage-depleted bone marrow cells sorted based on c-kit protein levels: c-kitHI (upper left), c-kitLO (middle left), and c-kitNEG (lower left). Hybridization was performed with the same probe used for Northern analysis. All antisense hybridizations were performed in tandem with sense control hybridizations (upper, middle, and lower right), and the silver grains visualized by dark field microscopy. Examples of cells positive for Hmgb3 mRNA are indicated by arrows. Original magnification, × 400.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Hmgb3 expression correlates with long-term repopulating ability.
Hmgb3 expression correlates with long-term repopulating ability. (A) Separation of Lin– c-kitHI Hmgb3-KI bone marrow cells into GFP+ and GFP– populations by flow cytometry (top). Sort gates were based on isotype controls (bottom). (B) Duplex RT-PCR performed on RNA isolated from sorted Hmgb3-KI bone marrow (BM) cells sorted into GFP+ and GFP– populations. RT-PCR performed without RT (–RT) was used for a negative control. β2-microglobulin cDNA amplification was used as an internal control. The amplicon sizes are 154 bp and 258 bp for Hmgb3 and β2-microglobulin, respectively. (C) Mean CFU-GM frequencies in Lin–, c-kit+, GFP– (n = 3, 202 colonies counted), and GFP+ (n = 3, 69 colonies counted) populations. Colony-forming assays were performed as described in “Materials and methods.” P values were determined by Student t test. (D) Representative hemoglobin analysis of competitive repopulation of Lin–, c-kitHI, GFP+ versus GFP– cells. Repopulations were performed with mixed doses of either Lin–, c-kitHI, GFP+ (n = 5) or Lin–, c-kitHI, GFP– (n = 5) and 107 C57BL/6 bone marrow as described in “Materials and methods.” 129/SvJ and C57BL/6 hemoglobin served as assay controls. The βMin, βMaj, (129/SvJ) and βS (C57BL/6) hemoglobins are indicated by black arrows. The average erythroid repopulation by Hmgb3-KI bone marrow (determined as percentage of total Hb that is of 129/SvJ genetic origin) for each population is underneath their respective samples accompanied by the standard deviation. Hb levels were quantified by densitometry.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Structure of the Hmgb3 gene and protein.
Structure of the Hmgb3 gene and protein. (A) Structure of Hmgb3 gene locus, mRNA, and protein. Top: schematic diagram of Hmgb3 gene locus (based on Vaccari et al).4 The entire locus is 4.4 kb flanked by PstI sites (P). There are 5 exons: white boxes represent untranslated regions, gray boxes represent translated regions. Bottom: Hmgb3 mRNA. The transcript is 1.5-kb long. Hmgb3 shares a highly conserved DNA binding domain with Hmgb1 and 2 (red bars). Green boxes represent putative nuclear localization signals based on Hmgb1 and 2 homology. The yellow bar represents the probe used for Northern blotting and in situ hybridization. (B) Nuclear localization of Hmgb3. Upper: constructs used to transfect 32D cells. Hmgb3 cDNA is fused in frame with the GFP gene. Lower: fluorescent microscopy of transfected 32D cells. DAPI staining was used to identify nuclei (blue). Green signals indicate Hmgb3-GFP protein. Some examples of overlapping signals are indicated by white arrows. Original magnification, × 400.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

5. Generation and characterization of Hmgb3 knock-in mice.
Generation and characterization of Hmgb3 knock-in mice. (A) Generation of Hmgb3 knock-in (Hmgb3-KI) mice by homologous recombination. Upper: diagram of Hmgb3 gene locus. Middle: targeting construct. An IRES-GFP cassette is inserted into the 3′ untranslated region of the Hmgb3 gene locus. Lower: Hmgb3 gene locus after recombination. (B) Screening of targeted TC1 ES cell clones. Representative Southern blot analysis of genomic DNA isolated from prospective clones digested with EcoRI. The wild-type Hmgb3 locus (+/Y) is contained on a 7.2-kb EcoRI fragment. Homologous recombination (GFP/Y) results in a hemizygous 8.9-kb EcoRI fragment. (C) Expression of Hmgb3-IRES GFP in Hmgb3-KI hematopoietic tissues. Representative results of FACS analysis performed on thymus, spleen, and bone marrow cells isolated from male Hmgb3-KI mice. Cells were stained with antibodies as described in “Materials and methods.” Upper panel: GFP expression in CD4-stained thymocytes, CD8-stained thymocytes, B220-stained splenocytes, and Ter119-stained bone marrow cells isolated from Hmgb3-KI mice. All other samples were negative for GFP expression. Lower panel: respective isotype controls.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Common lymphoid progenitors express Hmgb3.
Common lymphoid progenitors express Hmgb3. (A) Isolation of GFP+ and GFP– common lymphoid progenitors by flow cytometry. Upper: Lin– bone marrow isolated from Hmgb3-KI mice. Middle: c-kit and Sca-1 expression in Hmgb3-KI Lin– bone marrow (left). Cells positive for c-kit and Sca-1 were selected based on isotype staining of littermate control Lin– bone marrow cells (right). Lower: IL-7Rα and GFP profiles of c-kit+/Sca-1+ cells (left). Cells were separated into IL-7Rα+ and GFP+/– populations based on isotype staining of littermate control Lin– c-kit+, Sca-1+ bone marrow cells (Right). (B) Mean CFU–pre-B cell frequency in GFP– (n = 8, 66 colonies counted) and GFP+ (n = 7, 294 colonies counted) CLP populations. Values were determined by scoring pre–B cell colonies per 500 cells cultured. P values were determined by Student t test.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Analysis of Hmgb3 expression in myeloid progenitors (MP).
Analysis of Hmgb3 expression in myeloid progenitors (MP). (A) Isolation of myeloid progenitors by flow cytometry. Upper: Lin– bone marrow isolated from Hmgb3-KI mice. Middle: c-kit and Sca-1/IL-7Rα expression in Hmgb3-KI Lin– bone marrow. Cells positive for c-kit and negative for Sca-1/IL-7Rα were selected based on isotype controls (not shown). Lower: c-kit+/Sca-1 and IL-7Rα– cells were stained with either anti-FcγRII/III or CD34 monoclonal antibodies as described in “Materials and methods.” Left: FcγRII/III and GFP expression in Hmgb3-KI Lin– c-kit+/(Sca-1/IL-7Rα)– cells. Right: CD34 and GFP expression in Hmgb3-KI Lin– c-kit+/(Sca-1/IL-7Rα)– cells. Sort gates were drawn based on isotype controls (not shown). (B) Mean CFU-GM, BFU-E, and CFU-GEMM frequency in FcγRII/IIILO GFP– (n = 6) and GFP+ (n = 6) MP populations. Values were determined by scoring CFU-GM, BFU-E, and CFU-GEMM colonies per 500 cells cultured. P values were determined by Student t test. (C) Mean CFU-GM, BFU-E, and CFU-GEMM frequency in CD34+ GFP– (n = 5) and GFP+ (n = 4) MP populations. Values were determined by scoring CFU-GM, BFU-E, and CFU-GEMM colonies per 500 cells cultured. P values were determined by Student t test.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology

8. Overexpression of Hmgb3 impairs myeloid and B-lymphocyte differentiation.
Overexpression of Hmgb3 impairs myeloid and B-lymphocyte differentiation. (A) Diagram of Hmgb3-IRES-GFP retroviral vector and the control MSCV MGirL22Y IRES-GFP vector. (B) Mean CFU-C numbers per 1 × 105 cells plated for control IRES-GFP vector GFP– (n = 4; 223 colonies counted) and GFP+ (n = 3; 150 colonies counted) populations and for Hmgb3-IRES-GFP GFP– (n = 6; 424 colonies counted) and GFP+ (n = 6; 0 colonies counted) populations. (C) Representative FACS analysis of peripheral blood from mice who received transplants of either control IRES-GFP vector-transduced marrow (top: n = 8) or Hmgb3-IRES-GFP–transduced bone marrow (bottom: n = 10). Thymocytes, splenocytes, and bone marrow cells were analyzed 16 weeks after transplantation for presence of GFP in T cells, B cells, granulocytes/monocytes/macrophages, and erythroid cells, respectively. Quadrants were drawn based on isotype controls similar to those in Figure 3C. (D) Representative Southern blot analysis of genomic DNA isolated from bone marrow (M), thymus (T), and spleen (S) tissues from control GFP and Hmgb3-IRES-GFP mice 16-weeks after transplantation. 10 μg DNA were digested with EcoRI and loaded into each lane. Hybridization was performed with a probe that recognizes the GFP gene.
Michael J. Nemeth et al. Blood 2003;102:
©2003 by American Society of Hematology