SummaryBackgroundPregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes.MethodsIn this multicentre, open-label, randomised controlled trial, we recruited women aged 18–40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527.FindingsBetween March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference −0·19%; 95% CI −0·34 to −0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most...

Basic Krü ppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators.

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The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebp␣ promoter. We find that C/ebp␣ is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebp␣ promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.

Transcription factors that associate with DNA sequences in promoters and enhancers often recruit co-regulators that modulate their activity. Many of these co-regulators have intrinsic enzymatic activity and influence gene expression by modifying chromatin and altering its structure. Recently, a new family of co-repressors, the C-terminal binding proteins, has been described. These proteins recognize Pro-X-Asp-Leu-Ser (PXDLS) motifs in DNA-binding proteins and function as transcriptional co-repressors in Drosophila, Xenopus and mammals. The precise mechanisms by which they influence transcription are still under investigation. CtBP proteins dimerize and can contact histone deacetylases; hence they may operate by linking deacetylases to DNA-bound factors. But it appears that CtBP proteins also have intrinsic enzymatic activity. They have significant homology to D-isomer-specific 2-hydroxy acid dehydrogenases, and remarkably one family member, rat CtBP, has been shown to have a second role, functioning as an acyl transferase in Golgi maintenance. These observations raise the possibility that CtBP proteins might regulate gene expression directly by means of their enzymatic activities, in addition to serving as simple bridging proteins. Supplementary material for this article can be found on the BioEssays homepage at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/v23_8.684.

CACCC-boxes are recognised by transcription factors of the Sp/Krüppel-like Factor (Sp1/KLF) family. Here we describe one member of this family, KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Krüppel-like zinc fingers, but also has limited homology to another family member, KLF3/Basic Krüppel-like Factor (KLF3/BKLF), in its N-terminus. Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP). We demonstrate that the KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. We show that the N-terminus of KLF8 functions as a repression domain and that its activity relies on the integrity of the CtBP recognition motif. We demonstrate that the zinc fingers of KLF8 recognize CACCC elements in DNA and that full-length KLF8 can repress a CACCC-dependent promoter. Finally we determine that KLF8 is broadly expressed in human tissues. These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription.

We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.