Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Activation of the RhoB signaling pathway by thyroid hormone receptor β in thyroid cancer cells.

Thyroid hormone receptor (TR) mediates the crucial effects of the thyroid hormone (T3) on cellular growth, development, and differentiation. Decreased expression or inactivating somatic mutations of TRs have been found in human cancers of the liver, breast, lung, and thyroid. The mechanisms of TR-associated carcinogenesis are still not clear. To establish the function of TRβ in thyroid cancer cell proliferation, we constructed a recombinant adenovirus vector, AdTRβ, which expresses human TRβ1 cDNA. Thyroid cancer cell lines in which TRβ protein levels were significantly decreased as compared to intact thyroid tissues were infected with AdTRβ and the function of TRβ on cell proliferation and migration was analyzed. Ligand-bound TRβ induced HDAC1 and HDAC3 dissociation from, and histone acetylation associated with the RhoB promoter and enhanced the expression of RhoB mRNA and protein. In AdTRβ-infected cells, T3 and farnesyl transferase inhibitor (FTI)-treatment induced the distribution of RhoB on the cell membrane and enhanced the abundance of active GTP-bound RhoB. This RhoB protein led to p21-associated cell-cycle arrest in the G0/G1 phase, following inhibition of cell proliferation and invasion. Conversely, lowering cellular RhoB by small interfering RNA knockdown in AdTRβ-infected cells led to downregulation of p21 and inhibited cell-cycle arrest. The growth of BHP18-21v tumor xenografts in vivo was significantly inhibited by AdTRβ injection with FTIs-treatment, as compared to control virus-injected tumors. This novel signaling pathway triggered by ligand-bound TRβ provides insight into possible mechanisms of proliferation and invasion of thyroid cancer and may provide new therapeutic targets for thyroid cancers

TRβ infection–induced p21 expression and Go/G1 growth arrest.

<p>(A–C) Western blot analysis of the expression level of p21 or of the phosphorylated Rb protein in AdTRβ-infected BHP18-21v (A), FRO (B), or WRO (C) cells that were exposed to 30 nM of T3 alone for 12 h or to 30 nM T3 plus 5 µM of FTI for 12 h, with or without siRNA knockdown of RhoB as indicated. Tubulin was blotted as a loading control (bottom panels). (D–F) The percentage of cells in the G₀/G₁, S, or G₂/M phase was calculated by using ModFitLT version 3.1. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05.</p

Effects of ligand-bound TRβ on cancer cell proliferation and migration.

<p>(A–C) Cell proliferation assay. BHP18-21v (A), FRO (B) or WRO (C) cells were incubated in T3-depleted medium for 24 h and were then infected with 30 MOI of AdTRβ. The cells were then incubated in medium with T3 and/or FTI for an additional 24 or 72 h. Relative cell numbers were determined by arbitrarily setting the value for control cultures incubated in T3-free medium to 1. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05. (D–F) AdTRβ-infected BHP18-21v (D), FRO (E) or WRO (C) cells were seeded in the upper compartment of a migration chamber. After 24 h incubation, cells that had migrated to the lower surface were stained and quantified using fluorescence signals. Relative signals were determined by arbitrarily setting the value for control cultures incubated in T3-free medium to 1. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05.</p

Effects of ligand-bound TRβ and FTI on BHP18-21v xenografts.

<p>A. Relative tumor volume of AdTRβ-, AdLacZ-, or AdTRβPV treated xenografts over 22 days following viral injection was calculated by the formula V = 1/2 (length×width²). All mice were intraperitoneally injected with 100 mg/kg/body weight of FTI at each time points of viral injection. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05. B. Serum free T3 and TSH levels of adenovirus-injected and FTI-treated mice were analyzed by ELISA. The data points represent means ± S.D. (<i>n</i>  = 6). C. RhoB and p21 levels in the tumors were visualized with anti-RhoB and anti-p21 antibodies, respectively, and Alexa Fluor 555-conjugated second antibody (red); transfected TRβ was visualized with an anti-TRβ antibody and an Alexa Fluor 488-conjugated secondary antibody (green). Scale bars, 10 µm. D. Ki-67-positive cells are indicated in AdLacZ (a)- or AdTRβ (b)- treated xenografts on day 30. Scale bars, 50 µm. E. The Ki-67 proliferation indices (MIB-1 indices) of the xenografts are shown as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05. F. Cleaved caspase 3-positive cells are indicated in AdLacZ and FTI (a) or AdTRβ and FTI (b)- treated xenografts on day 30. Scale bars, 20 µm.</p

Ligand-bound TRβ binds the RhoB promoter and upregulates transcriptional activation.

<p>(A–C) RhoB promoter-Luciferase reporter assays. Adenovirus-infected BHP18-211v (A), FRO (B), or WRO (C) cells were transfected with 1 µg of a RhoB promoter-Luciferase reporter plasmid and were incubated for an additional 24 h in the absence or presence of 30 nM T3. Relative promoter activities (Luciferase activity) were determined by arbitrarily setting the value for control virus-infected cells incubated in T3-free medium to 1. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05. (D–F) ChIP assay using AdTRβ-infected BHP18-211v (D), FRO (E), or WRO (F) cells treated with or without 30 nM T3. Antibodies that recognize histone3 (H3), acetylated H3, HDAC1, HDAC3 or normal rabbit IgG were used for immunoprecipitation of the chromatin. Input indicates 10% of the chromatin. PCR products were separated on a 2% of agarose gel.</p

Expression of RhoB in AdTRβ-infected thyroid cancer cells.

<p>Expression levels of RhoB mRNA (A–C) or protein (D–F) in AdTRβ-infected, control AdLacZ-infected, or non-infected thyroid cancer cell lines exposed to 30 nM of T3 for 0, 6, 12 or 24 h as indicated. (A–C)The expression of RhoB and 18S mRNA was determined using real-time RT-PCR with 100 ng of cDNA. Relative mRNA expression levels were determined by arbitrarily setting the value for control virus-infected cells incubated in T3-free medium to 1. Data are expressed as means ± S.D. (<i>n</i>  = 6). *, <i>p</i><0.05. (D–F) Western blot analysis of 20 µg of protein lysates of the cells was performed using antibodies against RhoB (upper panel) or tubulin (lower panel), which was used as a loading control.</p