Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Western blot analysis of Runx1and Runx2 in WT and Runx1KO C2C12 relative to β-actin. (B) Representative phase contrast microscopy images of WT and Runx1KO C2C12 cells in growth medium (GM) or after 6 d in differentiation medium (DM). (C) Quantification of fusion index of WT and Runx1KO C2C12 cells after 6 days in differentiation medium. ***p<0.001. Data present as means ± SEM. n=3 for each group. (D) Differential gene expression analysis reveal differences in GM (D0) and DM (D1,2,6). The affected genes in each group/day are displayed here and in Supplemental Table S1. (E) Heat maps of the changes in 368 transcription factors: above the clusters, relative Log2 fold change in each column is shown. Below the clusters, the relative abundance (LogCPM) is shown. Members of the Notch pathway, Ets, MRF gene families are indicated above their relative position. (F) GO terms of the up- and downregulated genes in Runx1KO. Details in Supplemental Table S1.

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Western Blot, Microscopy, Expressing

Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Runx1KO C2C12 cells were transfected with 200ng Runx1 or Runx2 plasmids. After 24 hr, cells were induced to differentiate for 6 days followed by immunofluorescence staining for MHC (red) and DNA (Hoechst, blue). (B) Quantification of fusion index and of (C) MHC+ cell lengths in experiments shown in A. (D) Schematics of Runx1 and Runx2 chimera proteins. (E) Quantification of fusion index (F) and MHC+ cell lengths in experiments shown in G; (G) Runx1KO C2C12 cells were transfected with 200ng of five different chimera plasmids numbered in D. 24 h for after transfection, cells were induced to differentiate for 6 days followed by immunofluorescence staining for MHC (red) and DNA (Hoechst, Blue). Data presented as means ± SEM. n=3 for each group. ***p<0.001. n.s., not significant (Scale bars: 150μm.).

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

doi: 10.1101/2023.11.21.568117

Figure Lengend Snippet: (A) Western blots from co-immunoprecipitation experiments in C2C12 cells transfected with Myc tagged Runx proteins and Flag-tagged Etv4. (B) Venn diagram of ATACseq peaks from C2C12 or Runx1KO cells. Most peaks are shared, 9,318 peaks that are only found to C2C12 and 2,607 peaks are only found in Runx1KO C2C12 cells. (C) Heatmaps showing ATACseq reads from WT or Runx1KO C2C12 cells mapped onto these 3 classes of ATACseq peaks (WT specific, Shared, and Runx1KO specific). (D) Graph displaying the -log pValue of transcription factor motif enrichment as determined by HOMER. Runx1 motifs (blue) are more highly enriched in WT C2C12 cells, whereas Mef2 motifs (red MADS-box) are more enriched in Runx1KO C2C12. The inset shows that AP1 motifs (purple) are similarly enriched in both WT and C2C12 cells as expected for a global enhancer binding factor. (E) Bar graph showing enrichment for the Ets:Runx composite motif in Runx1 ChIP data from myoblasts or T-ALL cells, less enrichment seen in Runx2 ChIP from preosteoblasts. The composite motif enrichment in each dataset is normalized to enrichment rate of the Runx-only motif in the respective ChIP from each cell types.

Article Snippet: The mouse Runx2 plasmid pCMV-Flag-mRunx2 was purchased from Origene.

Techniques: Western Blot, Immunoprecipitation, Transfection, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Zfp521 is a physiological antagonist of Runx2 in vivo. (A) At postnatal day 0 (P0), Zfp521 +/− mice present a modestly elongated clavicle compared with Wt mice. Runx2 +/− mice exhibit a very severe hypoplasia of the clavicle, which is mitigated in Runx2 +/− ;Zfp521 +/− mice (arrows). (B) Compared with Wt mice, Zfp521 +/− mice demonstrate a more strongly mineralized midportion of the hyoid bone. The delayed mineralization of the midportion of the hyoid bone in Runx2 +/− mice is rescued in Runx2 +/− ;Zfp521 +/− mice (arrows). (C) A progressively severe hypoplasia of the clavicle was seen in postnatal day 2 (P2) Zfp521 Tg mice, Runx2 +/− mice, and Zfp521 Tg ;Runx2 +/− mice (arrows). (D) The midportion of the hyoid bones of P2 Zfp521 Tg mice, Runx2 +/− mice, and Zfp521 Tg ;Runx2 +/− mice show a progressive decrease in size and mineralization (arrows). (E) The calvaria of postnatal day 25 (P25) Zfp521 Tg mice were similar to the calvaria of Wt mice. Runx2 +/− mice exhibited open anterior and posterior fontanelles and wide cranial sutures, which were strikingly enhanced in Zfp521 Tg ;Runx2 +/− mice (arrows). Skeletal structures were measured. Data are mean ± SEM. *, P < 0.05 versus Wt . # , P < 0.05 versus Runx2 +/− . Littermates were analyzed as controls. Results are representative of six to eight animals. Bars, 1 mm.

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: In Vivo

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Zfp521 inhibits early stage osteoblast differentiation and increases the number of osteoprogenitors. (A) Gene expression analysis revealed that Runx2 and Osx mRNA expression was significantly increased in Zfp521 +/− neonatal calvarial osteoblasts compared with Wt mice. (B) Runx2 and Osx mRNA expression was significantly decreased in calvarial osteoblasts isolated from Zfp521 Tg mice. (C) Immunostaining and immunoblot (IB) analysis showed reduced Runx2 expression in calvarial osteoblasts from Zfp521 Tg mice and Wt littermates. Nuclei were stained with ToPro. Results are representative of three independent experiments. Bars, 10 µm. (D) Cultured calvarial cells from Zfp521 Tg mice and control littermates were stained for ALP activity, or the cells were lysed, and ALP activity was determined colorimetrically. ALP activity was reduced in cells overexpressing Zfp521. Bars, 5 mm. (E) [ 3 H]Thymidine incorporation by calvarial osteoblasts from Zfp521 Tg mice and control littermates showed no changes in osteoblast proliferation. (F) Zfp521 Tg mice contained an increased number of colony-forming unit–fibroblast (CFU-F) and colony-forming unit–osteoblast (CFU-OB). All results are representative of three to eight animals. All data are shown as mean ± SEM. *, P < 0.05 versus Wt .

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Expressing, Isolation, Immunostaining, Western Blot, Staining, Cell Culture, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Structure–function analysis of the Zfp521–Runx2 interaction. (A) Runx2 was immunoprecipitated (IP) from lysate of Wt calvarial osteoblasts, and the immune complexes were immunoblotted (IB) for Zfp521 and Runx2. Nonimmune IgG was the immunoprecipitated control. Presence of Zfp521 and Runx2 in total cell lysate (TCL) is shown. (B) Wild-type (WT) Xpress-Runx2 and fragments of Runx2 were overexpressed in HEK-293 cells, and the binding to immobilized GST-Zfp521 was determined by pull-down (PD) followed by immunoblotting (top). The expression of Runx2 proteins was confirmed by immunoblotting of cell lysates (middle). The schematic drawing shows Runx2 domains (AD, QA, Runt, PST, and RD) and the truncated forms. The binding of each fragment is indicated. (C) Chelating zinc ions with either EDTA or phenanthroline abolished the interaction of Flag-Runx2 with HA-Zfp521. HA-Zfp521 was immunoprecipitated with anti-HA in HEK-293 cells, and the immune complex was immunoblotted for Flag-Runx2 using anti-Flag antibody. (D) HA-tagged fragments of Zfp521 containing five zinc fingers (ZFs) each were constructed as shown in the schematic drawing. Each fragment was overexpressed (second panel, cell lysate) along with Flag-Runx2 (third panel, cell lysate) in HEK-293 cells and subjected to coimmunoprecipitation (top). Runx2 bound to the fragments containing ZFs 6–10 and 26–30 (summarized next to schematic drawing). (E and F) Zinc-coordinating His residues in fragments ZF 6–10 (E) and ZF 26–30 (F) were randomly mutated to disrupt individual ZF conformation. HA-tagged Zfp521 fragments with complementary disabled ZFs (marked in gray in the schematic drawings) and Flag-Runx2 were overexpressed in HEK-293 cells. Cell lysates were blotted for HA (second panels) or Flag (third panels). Binding of the mutated fragments to Runx2 is indicated. (G) ZFs 6 and 26 were disabled in full-length Zfp521 (mu_Zfp521). Neither Wt Zfp521 nor mu_Zfp521 alone affected the 6×OSE2-luc activity. In contrast to Wt Zfp521, mu_Zfp521 only partially antagonized Runx2 activity. Actin was used as a loading control in A–F. All results are representative of three to five independent experiments. Data are mean ± SEM. *, P < 0.05 versus control. # , P < 0.05 versus Runx2.

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Expressing, Zinc-Fingers, Construct, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Zfp521 represses Runx2 activity in an HDAC3-dependent manner. (A) Pull-down (PD) of Flag-Runx2 was performed in the absence or presence of Flag-Zfp521 using biotinylated oligonucleotides (Oligo; wild type [Wt] or mutated [mu]) encoding Runx2 consensus sites (rat [OG2] or mouse [OSE2] Osteocalcin promoter). Oligonucleotide-bound Flag-Runx2 was immunoblotted (IB) using anti-Flag antibody. HEK-293 cell lysates were analyzed by immunoblotting for overexpressed Flag-Runx2 and Flag-Zfp521. (B) Zfp521 prevented Runx2 from activating the 6×OSE2-luc reporter in ROS cells. The HDAC inhibitor trichostatin A (TSA) had little effect on the reporter alone or Runx2-induced activity but abolished Zfp521’s repression of Runx2-induced activity. (C) Δ13Zfp521 repressed the Runx2-driven 6×OSE2-luc reporter activity in ROS cells as well as full-length Zfp521. (D) Zfp521 fully reversed the Runx2-mediated 6×OSE2-luc reporter gene activation in Wt ROS cells (ROS17/2.8) and in ROS cells overexpressing a dominant-negative form of HDAC5 (ROS-H14). (E) Depleting endogenous HDAC3 using shRNA abolished the repression of Runx2-induced 6×OSE2-luc reporter gene activity by Zfp521. (F) Anti-HDAC3 coimmunoprecipitates (IPs) Zfp521 and HDAC3 from Wt calvarial osteoblast lysate. Nonimmune IgG was used as a control. HDAC3 is present in total cell lysate (TCL) as determined by immunoblotting. (G) Flag-HDAC3 was immunoprecipitated with anti-HDAC3 in ROS cells, and the immune complex was immunoblotted for Flag-Runx2 with anti-Flag antibody. (H) Endogenous Runx2 was immunoprecipitated from calvarial osteoblasts from Wt mice, or mice germline was deleted of Zfp521 (−/−), and endogenous HDAC3 and Runx2 were detected by immunoblotting. The interaction between Runx2 and HDAC3 was greatly reduced in osteoblasts lacking Zfp521 . Actin was used as loading control in F–H. In B–E, results are shown as mean ± SEM. *, P < 0.05 versus control. # , P < 0.05 versus Runx2. P-values are representative of three to five independent experiments.

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Activity Assay, Western Blot, Activation Assay, Dominant Negative Mutation, shRNA, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Zfp521 reverses the low bone mass in Runx2 Tg mice. Phenotypic characterization of 5-wk-old Wt , Zfp521 Tg , Runx2 Tg , and Zfp521 Tg ;Runx2 Tg female mice is shown. (A) Histomorphometric analysis of the trabecular bone in proximal tibiae (bone volume/tissue volume [BV/TV], bone formation rate/tissue volume [BFR/TV], mineral apposition rate [MAR], trabecular number [Tb.N], trabecular separation [Tb.Sp], number of osteoblasts/bone perimeter [N.Ob/B.Pm], and number of osteoclasts/bone perimeter [N.Oc/B.Pm]) demonstrates the normalization of the low bone mass of Runx2 Tg mice by Zfp521. (B) Analysis of the cortical thickness (Ct.Th) in the tibia midshaft. Cortical mineral apposition rate (Ct.MAR) was determined by calcein/democycline double labeling (double arrows). Bars, 20 µm. (C) Cortical porosity (Ct. porosity) was quantified using Sirius red–stained decalcified paraffin-embedded cortical sections. Bars, 100 µm. (D) Tartrate-resistant acid phosphatase staining of decalcified paraffin-embedded cortical sections shows increased numbers of osteoclasts and resorption in the cortical bone of Runx2 Tg mice compared with Zfp521 Tg and Wt mice, which was normalized by Zfp521. Ct.Ar., cortical area. Bars, 100 µm. Data are mean ± SEM. n = 4–6 mice per group. *, P < 0.05 versus Wt . # , P < 0.05 versus Runx2 Tg .

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Labeling, Staining

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Zfp521 relieves the Runx2-induced block of osteoblast maturation. (A–C) Expression of osteopontin (A), osteocalcin (B), and dentin matrix protein 1 (Dmp-1; C) was determined by in situ hybridization of decalcified tibiae. In the trabecular bone of Runx2 Tg mice, partially differentiated Osteopontin-expressing osteoblasts were increased, and mature Osteocalcin-expressing osteoblasts were decreased compared with Zfp521 Tg and Wt littermates, as were Dmp-1–expressing osteocytes in the cortical bone. Overexpressing Zfp521 normalized the number of Osteopontin-positive cells and restored the abundance of mature osteoblasts and osteocytes in Zfp521 Tg ;Runx2 Tg mice. The number of osteocytes in the cortical bone was determined per bone volume (Ocy.N./BV). Bars, 100 µm. (D) Changes in the number of osteocytes were confirmed by quantifying Dmp-1 mRNA expression in the cortical bones of the same animals. (E) The increased Rankl/Opg ratio in 5-wk-old female Runx2 Tg mice was normalized in Zfp521 Tg ;Runx2 Tg animals. (F) Radiograms of the right tibia of 12-wk-old male mice. Runx2 Tg mice had a mean of two midshaft fractures in hind-limb long bones (fracture rate). The fracture rate was reduced by 80% in Zfp521 Tg ;Runx2 Tg animals. Bars, 2.5 mm. All data are mean ± SEM. n = 4–6 mice per group. *, P < 0.05 versus Wt . # , P < 0.05 versus Runx2 Tg .

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Blocking Assay, Expressing, In Situ Hybridization

Journal: The Journal of Cell Biology

Article Title: Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

doi: 10.1083/jcb.201009107

Figure Lengend Snippet: Model of the regulation of osteoblast differentiation by Zfp521. Zfp521 interacts with HDAC3 and Runx2, stabilizing the association of these two factors. This corepressor complex represses target gene transcription. Runx2 is required for entry of progenitor cells to the osteoblast lineage. Zfp521 antagonizes this early positive effect of Runx2, leading to decreased osteoblast differentiation and an increased number of progenitors. Committed osteoblasts express Rankl and stimulate osteoclastogenesis. Runx2 antagonizes later stages of osteoblast differentiation, blocking osteoblast maturation. This block leads to an increase of Rankl-expressing immature osteoblasts and therefore to an increase in bone resorption, a decrease in the abundance of terminally differentiated osteoblasts and osteocytes, and ultimately fractures in Runx2 Tg mice. Zfp521 also antagonizes this later negative effect of Runx2, thereby removing the maturational block and rescuing the low bone mass of Runx2 Tg mice. The high bone mass phenotype of Zfp521 Tg mice suggests that Zfp521 exerts a greater effect on Runx2 activity at the later stage of osteoblast differentiation.

Article Snippet: Several fragments of Runx2 encoding individual domains or small groups of domains were amplified from the pCMV-Flag-Runx2 construct by PCR and subcloned in the pcDNA3.1 vector (Invitrogen).

Techniques: Blocking Assay, Expressing, Activity Assay