Abstract
Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (betaMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct betaMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair betaMyHC promoter fused to chloramphenicol acetyltransferase (beta293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the betaMyHC negative regulatory element (-332 to -300; betaNRE) within transgene beta350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the betaNRE sense strand (dbetaNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the betaNRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated betaMyHC promoter elements transcriptionally regulate betaMyHC transgene expression in response to two diverse modes of neuromuscular activity.
Highlights
Our previous transgenic analyses revealed that a 600base pair -myosin heavy chain (MyHC) promoter conferred mechanical overload (MOV) and non-weightbearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene
Do MOV and NWB activity direct MyHC gene transcription via the same or distinct cisacting element(s)? Second, do MOV and/or NWB element(s) serve an independent or overlapping role in specifying fiber type-specific expression? Third, does the NRE contribute to the decrease in MyHC expression seen under NWB conditions in vivo? As an initial step toward resolving these critical regulatory mechanism(s), we have set out to delimit a MyHC gene promoter region that is responsive to NWB activity by generating transgenic lines harboring transgenes composed of either 293 or 350 bp of the human MyHC gene proximal promoter region (293, 350) fused to the 5Ј-end of the CAT gene
Notable findings resulting from this transgenic study are that the DNA regulatory sequence(s) that mediate MyHC gene expression in response to either MOV or NWB activity are segregated to different regions within the proximal promoter of the MyHC gene and that a negative regulatory element (dNRE-S) that interacts with two unidentified protein(s), may play an in vivo role in NWB regulation of MyHC
Summary
MyHC, myosin heavy chain; MyHC, -myosin heavy chain; NRE, MyHC negative regulatory element; dNRE, distal region of NRE; dNRE-S, distal region of the NRE sense strand; CNBP, cellular nucleic acid-binding protein; YB-1 and -3, Y-box-binding factor 1 and 3, respectively; MOV, mechanical overload; NWB, non-weight-bearing; bp, base pair(s); CS, control-soleus; NWB-S, NWB-soleus; CP, control-plantaris; MOV-P, MOV-plantaris; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay. It was shown that additional sequences located upstream of nucleotide Ϫ600 are required for high levels of muscle-specific MyHC transgene expression and that these element(s) can compensate for the loss of any one or two control region elements [15, 16] These in vivo findings indicate that complex combinatorial interactions between distal and proximal DNA regulatory elements are required for high levels of striated muscle-specific expression. Notable findings resulting from this transgenic study are that the DNA regulatory sequence(s) that mediate MyHC gene expression in response to either MOV or NWB activity are segregated to different regions within the proximal promoter of the MyHC gene and that a negative regulatory element (dNRE-S) that interacts with two unidentified protein(s), may play an in vivo role in NWB regulation of MyHC
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