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The zebrafish as a model system for analyzing mammalian and native ?-crystallin promoter function.

ABSTRACT: Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish ?-crystallin promoter function, how it compares to that of mammals, or whether mammalian ?-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of ?-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish ?-crystallins. Our data showed that mouse and zebrafish ?A-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse ?B-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish ?B-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the ?Bb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish ?A increased between 1 and 2 days post fertilization (dpf), ?Ba increased between 4 and 5 dpf, but ?Bb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect ?A, aBa, and ?Bb peptides in digests of zebrafish embryos. In whole embryos, ?A-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. ?Ba was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no ?Bb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian ?-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

SUBMITTER: Posner M

PROVIDER: S-EPMC5708185 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The zebrafish as a model system for analyzing mammalian and native <i>α</i>-crystallin promoter function.

Posner Mason M Murray Kelly L KL McDonald Matthew S MS Eighinger Hayden H Andrew Brandon B Drossman Amy A Haley Zachary Z Nussbaum Justin J David Larry L LL Lampi Kirsten J KJ

PeerJ 20171127

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafis ...[more]

PMID: 29201567

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Project description:Extracytoplasmic function (ECF) ? factors are the largest and the most diverse group of alternative ? factors, but their mechanisms of transcription are poorly studied. This subfamily is considered to exhibit a rigid promoter structure and an absence of mixing and matching; both -35 and -10 elements are considered necessary for initiating transcription. This paradigm, however, is based on very limited data, which bias the analysis of diverse ECF ? subgroups. Here we investigate DNA and protein recognition motifs involved in ECF ? factor transcription by a computational analysis of canonical ECF subfamily members, much less studied ECF ? subgroups, and the group outliers, obtained from recently sequenced bacteriophages. The analysis identifies an extended -10 element in promoters for phage ECF ? factors; a comparison with bacterial ? factors points to a putative 6-amino-acid motif just C-terminal of domain ?2, which is responsible for the interaction with the identified extension of the -10 element. Interestingly, a similar protein motif is found C-terminal of domain ?2 in canonical ECF ? factors, at a position where it is expected to interact with a conserved motif further upstream of the -10 element. Moreover, the phiEco32 ECF ? factor lacks a recognizable -35 element and ?4 domain, which we identify in a homologous phage, 7-11, indicating that the extended -10 element can compensate for the lack of -35 element interactions. Overall, the results reveal greater flexibility in promoter recognition by ECF ? factors than previously recognized and raise the possibility that mixing and matching also apply to this group, a notion that remains to be biochemically tested.ECF ? factors are the most numerous group of alternative ? factors but have been little studied. Their promoter recognition mechanisms are obscured by the large diversity within the ECF ? factor group and the limited similarity with the well-studied housekeeping ? factors. Here we extensively compare bacterial and bacteriophage ECF ? factors and their promoters in order to infer DNA and protein recognition motifs involved in transcription initiation. We predict a more flexible promoter structure than is recognized by the current paradigm, which assumes rigidness, and propose that ECF ? promoter elements may complement (mix and match with) each other's strengths. These results warrant the refocusing of research efforts from the well-studied housekeeping ? factors toward the physiologically highly important, but insufficiently understood, alternative ? factors.

Dataset Information

The zebrafish as a model system for analyzing mammalian and native ?-crystallin promoter function.

Publications

The zebrafish as a model system for analyzing mammalian and native <i>α</i>-crystallin promoter function.

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