Jiyue Zhu

A New Positive/Negative Selection Scheme for Precise BAC Recombineering

Recombineering technology allows the modification of large DNA constructs without using restriction enzymes, enabling the use of bacterial artificial chromosomes (BACs) in genetic engineering of animals and plants as well as in the studies of structures and functions of chromosomal elements in DNA replication and transcription. Here, we report a new selection scheme of BAC recombineering. A dual kanamycin and streptomycin selection marker was constructed using the kanamycin resistance gene and bacterial rpsL+ gene. Recombination cassettes generated using this dual marker was used to make precise modifications in BAC constructs in a two-step procedure without leaving behind any unwanted sequences. The dual marker was first inserted into the site of modifications by positive selection of kanamycin resistance. In the second step, the counter-selection of streptomycin sensitivity resulted in the replacement of the dual marker with intended modified sequences. This method of BAC modification worked as efficiently as the previously reported galK method and provided a faster and more cost-effective alternative to the galK method.

Chromatin and epigenetic regulation of the telomerase reverse transcriptase gene

Telomerase expression and telomere maintenance are critical for long-term cell proliferation and survival, and they play important roles in development, aging, and cancer. Cumulating evidence has indicated that regulation of the rate-limiting subunit of human telomerase reverse transcriptase gene (hTERT) is a complex process in normal cells and many cancer cells. In addition to a number of transcriptional activators and repressors, the chromatin environment and epigenetic status of the endogenous hTERT locus are also pivotal for its regulation in normal human somatic cells and in tumorigenesis.

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)‐like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self‐renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.—Mathew, R., Jia, W., Sharma, A., Zhao, Y., Clarke, L. E., Cheng, X., Wang, H., Salli, U., Vrana, K. E., Robertson, G. P., Zhu, J., Wang, S. Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J. 24, 2702–2715 (2010). www.fasebj.org

The hTERT Gene Is Embedded in a Nuclease-resistant Chromatin Domain

Normal human cells rarely undergo spontaneous immortalization. Given that ectopic expression of the human telomerase catalytic subunit hTERT leads to cellular immortalization, the endogenous hTERT gene is likely constitutively repressed. Hence, we have examined the chromatin structure of the native hTERT locus and the neighboring loci, CRR9 and Xtrp2, in normal human fibroblasts and a set of immortal lines. Using generalized DNase I sensitivity assays, we revealed that the entire hTERT gene was embedded in a chromatin domain that was as resistant to the nuclease as the well studied β-globin loci in both telomerase-positive and -negative cells. This condensed domain was at least 100 kb in size and contained the intergenic region 5′ to the hTERT gene and the downstream Xtrp2 locus. A transition from the nuclease-sensitive CRR9 locus to the condensed region appeared near the 3′-end of the CRR9 gene. hTERT transcription was associated with the appearance of a major DNase I-hypersensitive site positioned around the hTERT transcription start site and several minor hypersensitive sites. In telomerase-negative cells, the inhibition of histone deacetylases by trichostatin A led to the opening of this chromatin domain, accompanied by transcription from the hTERT gene but not the Xtrp2 gene. In contrast, the inhibition of protein synthesis by cycloheximide induced transcription from both the hTERT and Xtrp2 genes, indicating that histone deacetylases and labile factors coordinate to silence this chromosomal region. Taken together, our data suggest a novel mechanism of hTERT regulation at the chromatin level and have important implications for studying telomerase expression.

Dual roles of c-Myc in the regulation of hTERT gene

Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at -165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.

Differential repression of human and mouse TERT genes during cell differentiation

Differential regulation of telomerase reverse transcriptase (TERT) contributes to the distinct aging and tumorigenic processes in humans and mice. Here, we report that the hTERT gene was strongly repressed during differentiation of human cells, whereas modest mTERT expression was detected in terminally differentiated and post-mitotic cells. The stringent hTERT repression depended on the native chromatin environment because transiently transfected hTERT promoters were not repressed in differentiated cells. Conversely, the transiently transfected mTERT core promoter was repressed during cell differentiation, suggesting that the repression of mTERT promoter did not require its endogenous chromatin structures. To understand the mechanisms of this differential regulation, we examined chromatin structures of the endogenous TERT loci during cell differentiation. In both human and mouse cells, repression was accompanied by the loss of multiple DNase I hypersensitive sites at the TERT promoters and their upstream regions, revealing positions of potential regulatory elements. Interestingly, the hTERT locus was located within a nuclease-resistant chromatin domain in human cells, whereas a corresponding chromatin domain was not detected for the mTERT locus. Taken together, our study indicated that, unlike the repression of mTERT gene, the condensed native chromatin environment of hTERT locus was central to its silencing during cell differentiation.

Rearrangement of upstream sequences of the hTERT gene during cellular immortalization.

Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase‐negative (Tel−) and ‐positive (Tel+) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel+ cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel+ cell lines but not in their parental precrisis cells and Tel− immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel+ cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment.

A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo,we engineered mice with a 160‐kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a ReniUa luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species‐specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. Inaddition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1‐induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position‐independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.—Jia, W., Wang, S., Horner, J. W., Wang, N., Wang, H., Gunther, E. J., DePinho, R. A., Zhu, J. A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis. FASEB J. 25, 979–989 (2011). www.fasebj.org

Distinct and Temporal Roles of Nucleosomal Remodeling and Histone Deacetylation in the Repression of the hTERT Gene

Transcriptional silencing of the hTERT gene during HL60 cell differentiation was a biphasic process. The initial repression was accompanied by the loss of c-Myc binding and disappearance of a nucleosome-free region at the core promoter. The subsequent nucleosomal remodeling and histone modifications at the promoter stabilized this repression.