Molecular and Functional Analysis of the Utrophin Promoter

Abstract

Utrophin is a ubiquitously expressed cytoskeletal protein which is an important structural component of the mammalian neuromuscular junction. It shows extensive sequence similarity to dystrophin leading to postulation that utrophin may be able to compensate for the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients. In order to study the transcriptional control of utrophin expression including its regulation at the neuromuscular junction, and as a first step in the development of a potential DMD therapy, we have cloned the utrophin promoter region from human and mouse. The utrophin promoter is associated with a CpG island at the 5'-end of the gene, and sequence analysis of the 5'-UTR reveals several Sp1 binding sites and the absence of TATA or CAAT motifs. Transcription is initiated at one major and three minor sites. Using deletion constructs, we have defined an active promoter region of 155 bp. The first exon and 900 bp upstream display limited sequence conservation between human and mouse. The core sequence TTCCGG of the N box which regulates synaptic expression of other genes is also present and may be involved in regulating the specific expression of utrophin at the postsynaptic membrane. This study provides the basis for the understanding of the regulatory mechanism that controls utrophin expression and provides the data needed to develop methods for the upregulation of utrophin in DMD patients.

The artificial zinc finger coding gene ‘Jazz’ binds the utrophin promoter and activates transcription

“…As shown in Figure 1, Jazz was constructed to have the highest probability of targeting the DNA sequence (5Ј-GCT-GCT-GCG-3Ј) 'J' present both in the human and mouse utrophin gene promoter region. 32 The target 'J' has been chosen, since its DNA sequence is compatible with the appliance of the 'proposed code' and it is perfectly conserved between human and mouse utrophin promoters. Moreover, the DNA region in proximity of the J target includes conserved elements as 'N and E' boxes, suggesting the possibility of a favorable conformation of the chromatin in terms of transcription factors accessibility.…”

“…The pXP-Wt construct, previously described, 32 contains the XhoI-PstI DNA fragment, 316 bp long, of the human utrophin promoter (see diagram in Figure 6c) cloned in the pGL2 Basic vector (Promega). Mutations in the J target sequence of pXP-Wt construct were introduced using the Quick change mutagenesis kit (Stratagene, La Jolla, CA, USA) following the manufacturer's instructions and verified by DNA sequencing.…”

“…Jazz has been designed to recognize the target sequence 5Ј-GCT GCT GCG-3Ј (named 'J') present in the promoter region of the human and mouse utrophin genes. 32 Here we report on the DNA binding affinity/ specificity of the new artificial Jazz protein. Furthermore, we describe two artificial Jazz chimeric transcription factors: Gal4-Jazz and Sp1-Jazz, capable of driving transcription from the human utrophin promoter.…”

Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

“…The pPUBF utrophin promoter luciferase reporter construct contains 1.3-kb of human utrophin promoter-A sequence (Dennis et al, 1996), and it was a kind gift from Prof. Kay Davies (Oxford University). The pPUBF⌬N-box is a pPUBF derivative with a deleted N-box/EBS sequence, and it has been previously described by our laboratory (Khurana et al, 1999).…”

“…As shown in Figure 2Aii, incubation with U0126 caused ERF to preferentially localize in the nucleus. To demonstrate the effect of ERF on utrophin-A promoter activity, we used the pPUBF utrophin-A luciferase reporter construct containing the entire human utrophin promoter (Dennis et al, 1996), as well as the pUBF⌬N-box reporter construct that contains a deletion mutation of the N-box region (Khurana et al, 1999) (Figure 2Bi). Drosophila S2 cells were cotransfected with pPUBF and 1) expression constructs encoding GABP␣/␤ (Rosmarin et al, 1995) as a positive control or 2) ERF expression vector pSG5-ERF.…”